Multispecific Antigen-Binding Molecules for Cell Targeting and Uses Thereof

ABSTRACT

The present invention provides multispecific antigen-binding molecules that bind both a T-cell antigen (e.g., CD3) and a target antigen (e.g., a tumor associated antigen, a viral or bacterial antigen), and which include a single polypeptide chain that is multivalent (e.g., bivalent) with respect to T-cell antigen binding, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/993,721, filed Aug. 14, 2020, which claims the benefit under 35 USC §119(e) of U.S. Provisional Application Nos.: 62/887,411, filed Aug. 15,2019; 62/924,435, filed Oct. 22, 2019; 62/978,584, filed Feb. 19, 2020;and 63/057,824, filed Jul. 28, 2020, each of which is incorporatedherein by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 10606U502-Sequence.txt,created on Apr. 8, 2022 and containing 64,610 bytes.

FIELD OF THE INVENTION

The present invention relates to alternative formats for multivalentantigen-binding proteins, and methods of use thereof. The multivalentantigen-binding proteins, including bispecific and multispecificmolecules comprise a first polypeptide chain with both an N-terminal anda C-terminal antigen-binding domain that specifically binds a T-cellantigen (e.g., CD3), and a second polypeptide chain comprising at leastone antigen-binding domain that binds a target antigen (e.g., a tumorcell antigen).

BACKGROUND

Bispecific and multispecific antibodies and antigen-binding moleculesare known in the art (see, e.g., Brinkmann and Kontermann, MABS, 9(2):182-212, 2017). Among such known formats is the FcFc* (FIG. 1Astructure), a traditional bispecific antibody with Fab antigen-bindingdomains on either arm of the antibody and an Fc region with a modifiedCH3 domain that changes Protein A binding affinity to permit isolationof the heterodimer from the homodimeric impurities (Id. at p. 184, FIG.2, panel 7, last structure). This traditional bispecific antibody formathas been used to make bispecific antibodies in which one arm of theantibody targets a tumor cell antigen and the second arm targets aT-cell antigen, such as CD3. Another conventional format is theIgG-HC-scFv (FIG. 1B structure), a bispecific antibody in which twoN-terminal Fab domains bind a first antigen and two scFv domains linkedto the C-terminus of the Fc region bind a second antigen (Id. at p. 184,FIG. 2, panel 10, first structure). There is a need in the art for newand useful formats for bispecific or multispecific antigen-bindingmolecules that improve desired functionalities. Although Brinkmann etal. generically references the “building blocks” for the generation ofany homodimeric or heterodimeric antigen-binding molecule (p. 183, FIG.1), the possibilities are virtually infinite, and only those moleculesshown in FIG. 2 (p. 184) had reportedly been prepared. Moreover,Brinkmann doesn't contemplate specific antigen-binding domains,particularly a molecule comprising T-cell antigen binding domains atboth the N-terminus and the C-terminus of a single polypeptide chainforming part of a multispecific molecule.

BRIEF SUMMARY OF THE INVENTION

In general, the present invention provides multispecific antigen-bindingmolecules that bind both a T-cell antigen (TCA) (e.g., CD3) and a targetantigen (TA) (e.g., a tumor associated antigen, a viral or bacterialantigen), and which include a single polypeptide chain that ismultivalent (e.g., bivalent) with respect to T-cell antigen binding.

In one aspect, the present invention provides a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first antigen-bindingdomain that specifically binds a T cell antigen, (ii) a firstmultimerizing domain, and (iii) a second antigen-binding domain thatspecifically binds a T cell antigen; and (b) a second polypeptidecomprising, from N-terminus to C-terminus (i) a third antigen-bindingdomain that specifically binds a target antigen, and (ii) a secondmultimerizing domain, wherein the first and the second multimerizingdomains associate with one another to form the molecule.

In some embodiments, the second polypeptide further comprises a fourthantigen-binding domain at the C-terminus of the second multimerizingdomain. In some cases, the fourth antigen-binding domain specificallybinds a target antigen. In some cases, the third antigen-binding domainand the fourth antigen-binding domain specifically bind distinct targetantigens. In some cases, the distinct target antigens are expressed (orpresent) on the surface of the same cell. In some cases, the distincttarget antigens are expressed (or present) on the surface of differentcells. References, herein, to a target antigen expressed (or present) onthe surface of a cell include both a protein expressed by the cell thatis embedded in or spans the cell's membrane, and a peptide presented inthe context of the groove of a major histocompatibility complex (MHC)protein by the cell. In some cases, the third antigen-binding domain andthe fourth antigen-binding domain specifically bind the same targetantigen. In some embodiments, the fourth antigen-binding domainspecifically binds a T cell antigen. In some cases, the firstantigen-binding domain and the second antigen-binding domainspecifically bind the same T-cell antigen. In some cases, the firstantigen-binding domain and the second antigen-binding domainspecifically bind distinct T-cell antigens. In some embodiments, thefirst antigen-binding domain specifically binds a first T-cell antigenthat is a co-stimulatory molecule, and the second antigen-binding domainspecifically binds a second T-cell antigen that is a check-pointinhibitor. In some cases, the co-stimulatory molecule is CD28 and thecheck-point inhibitor is PD-1. In some cases, the first, second andfourth antigen-binding domains specifically bind the same T-cellantigen. In some cases, the first, second and fourth antigen-bindingdomains bind distinct T-cell antigens. In some cases, the first andfourth antigen-binding domains specifically bind the same T-cellantigen. In some cases, the second and fourth antigen-binding domainsspecifically bind the same T-cell antigen.

In various embodiments, one or more of the antigen-binding domains is aFab. In various embodiments, one or more of the antigen-binding domainsis a scFv. In some embodiments, the multispecific molecules contain bothFab and scFv antigen-binding domains. In some cases, the firstantigen-binding domain and the third antigen-binding domain are Fabs. Insome cases, the second antigen-binding domain is an scFv. In some cases,the fourth antigen-binding domain is an scFv. In some embodiments, thefirst, second and third antigen-binding domains are Fabs. In some cases,the first and third antigen-binding domains are Fab domains, and thesecond antigen-binding domain is an scFv domain. In some embodiments,the first, second, third and fourth antigen-binding domains are Fabs. Insome cases, the first, second, third and fourth antigen-binding domainsare Fab domains. In some cases, the first and third antigen-bindingdomains are Fab domains, and the second and fourth antigen-bindingdomains are scFv domains. In some cases, the first, second, third andfourth antigen-binding domains are Fab domains. In some cases, the firstand third antigen-binding domains are Fab domains, and the second andfourth antigen-binding domains are scFv domains. In some cases, thefirst, second, third and fourth antigen-binding domains are Fab domains.

In any embodiments in which the antigen-binding domain is an scFvdomain, the scFv domain may comprise a heavy chain variable region(HCVR) comprising a cysteine mutation at residue 44, and a light chainvariable region comprising a cysteine mutation at residue 100 (Kabatnumbering). In some cases, the scFv comprises a HCVR and a LCVR joinedtogether via a polypeptide linker of from 10 to 30 amino acids,optionally a (G4S)₄ linker. In some embodiments, the scFv is connectedto the C-terminus of the first and/or second multimerizing domain via alinker of from 5 to 25 amino acids, optionally a (G4S)₃ linker.

In some embodiments, the T cell antigen is a T cell receptor complexantigen (i.e., any of the protein subunits that make up the T cellreceptor complex). In some cases, the T cell antigen is CD3. In somecases, the T cell antigen is a co-stimulatory molecule or a check-pointinhibitor on a T cell. In some embodiments, the T cell antigen isselected from the group consisting of CD27, CD28, 4-1BB and PD-1. Insome embodiments, the T cell antigen is selected from the groupconsisting of CD3, CD27, CD28, 4-1BB and PD-1.

In some embodiments, the target antigen is a tumor-associated antigen.In some embodiments, the target antigen is a viral or bacterial antigen.In some embodiments, the target antigen is a fungal antigen or aparasite antigen.

In some embodiments, the first and second multimerizing domains areimmunoglobulin Fc domains. In some cases, the first multimerizing domainand the second multimerizing domain are human IgG1 or human IgG4 Fcdomains. In some cases, the first and second multimerizing domainscomprise an immunoglobulin hinge domain, a CH2 domain and a CH3 domainof a human IgG polypeptide (e.g., IgG1, IgG2, IgG3 or IgG4). In somecases, the first and second multimerizing domains comprise a hingedomain, a CH2 domain and a CH3 domain of a human IgG1 polypeptide. Insome cases, the first and second multimerizing domains comprise a hingedomain, a CH2 domain and a CH3 domain of a human IgG4 polypeptide. Insome embodiments, the first and second multimerizing domains associatewith one another via disulfide bonding.

In some embodiments, the first multimerizing domain or the secondmultimerizing domain comprises an amino acid substitution that reducesaffinity for Protein A binding compared to a wild-type Fc domain of thesame isotype. In some cases, the amino acid substitution comprises anH435R modification, or H435R and Y436F modifications (EU numbering). Insome cases, the first multimerizing domain comprises the H435R and Y436Fmodifications. In some cases, the second multimerizing domain comprisesthe H435R and Y436F modifications. In some embodiments, the firstpolypeptide, the second polypeptide, or both the first and the secondpolypeptides comprise a modified hinge domain that reduces bindingaffinity for an Fcγ receptor relative to a wild-type hinge domain of thesame isotype.

In another aspect, the present invention provides a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first Fab thatspecifically binds a T cell antigen, (ii) a first immunoglobulin Fcdomain, and (iii) a first scFv that specifically binds a T cell antigen;and (b) a second polypeptide comprising, from N-terminus to C-terminus(i) a second Fab that specifically binds a target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a second scFv that specificallybinds a target antigen, wherein the first and the second immunoglobulindomains associate with one another via disulfide bonding to form themolecule.

In some embodiments, the second Fab and the second scFv specificallybind distinct target antigens. In some cases, the distinct targetantigens are expressed on the surface of the same cell. In someembodiments, the second Fab and the second scFv specifically bind thesame target antigen.

In another aspect, the present invention provides a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first Fab thatspecifically binds a T cell antigen, (ii) a first immunoglobulin Fcdomain, and (iii) a second Fab that specifically binds a T cell antigen;and (b) a second polypeptide comprising, from N-terminus to C-terminus(i) a third Fab that specifically binds a target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a fourth Fab that specifically bindsa target antigen, wherein the first and the second immunoglobulindomains associate with one another via disulfide bonding to form themolecule.

In some embodiments, the third Fab and the fourth Fab specifically binddistinct target antigens. In some cases, the distinct target antigensare expressed on the surface of the same cell. In some embodiments, thethird Fab and the fourth Fab specifically bind the same target antigen.

In another aspect, the present invention provides a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first Fab thatspecifically binds a T cell antigen, (ii) a first immunoglobulin Fcdomain, and (iii) a first scFv that specifically binds a T cell antigen;and (b) a second polypeptide comprising, from N-terminus to C-terminus(i) a second Fab that specifically binds a target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a second scFv that specificallybinds a T cell antigen, wherein the first and the second immunoglobulindomains associate with one another via disulfide bonding to form themolecule.

In another aspect, the present invention provides a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first Fab thatspecifically binds a T cell antigen, (ii) a first immunoglobulin Fcdomain, and (iii) a second Fab that specifically binds a T cell antigen;and (b) a second polypeptide comprising, from N-terminus to C-terminus(i) a second Fab that specifically binds a target antigen, and (ii) asecond immunoglobulin Fc domain, wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule.

In various embodiments, such as any of those mentioned above or herein,the T cell antigen is a T cell receptor complex antigen (i.e., any ofthe protein subunits that make up the T cell receptor complex). In somecases, the T cell antigen is CD3. In some cases, the T cell antigen is aco-stimulatory molecule or a check-point inhibitor on a T cell. In someembodiments, the T cell antigen is selected from the group consisting ofCD27, CD28, 4-1BB and PD-1. In some embodiments, the T cell antigen isselected from the group consisting of CD3, CD27, CD28, 4-1BB and PD-1.

In various embodiments, such as any of those mentioned above or herein,the target antigen is a tumor-associated antigen. In some embodiments,the target antigen is a viral or bacterial antigen. In some embodiments,the target antigen is a fungal antigen or a parasite antigen.

In some embodiments, such as any of those mentioned above or herein, thefirst and second multimerizing domains are immunoglobulin Fc domains. Insome cases, the first multimerizing domain and the second multimerizingdomain are human IgG1 or human IgG4 Fc domains. In some cases, the firstand second multimerizing domains comprise an immunoglobulin hingedomain, a CH2 domain and a CH3 domain of a human IgG polypeptide (e.g.,IgG1, IgG2, IgG3 or IgG4). In some cases, the first and secondmultimerizing domains comprise a hinge domain, a CH2 domain and a CH3domain of a human IgG1 polypeptide. In some cases, the first and secondmultimerizing domains comprise a hinge domain, a CH2 domain and a CH3domain of a human IgG4 polypeptide. In some embodiments, the first andsecond multimerizing domains associate with one another via disulfidebonding.

In some embodiments, such as any of those mentioned above or herein, thefirst multimerizing domain or the second multimerizing domain comprisesan amino acid substitution that reduces affinity for Protein A bindingcompared to a wild-type Fc domain of the same isotype. In some cases,the amino acid substitution comprises an H435R modification, or H435Rand Y436F modifications (EU numbering). In some cases, the firstmultimerizing domain comprises the H435R and Y436F modifications. Insome cases, the second multimerizing domain comprises the H435R andY436F modifications. In some embodiments, the first polypeptide, thesecond polypeptide, or both the first and the second polypeptidescomprise a modified hinge domain that reduces binding affinity for anFcγ receptor relative to a wild-type hinge domain of the same isotype.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising any one of the multispecific molecules discussedabove or herein, and a pharmaceutically acceptable carrier or diluent.

In another aspect, the present invention provides a method of treatingcancer, comprising administering any one of the multispecific moleculesdiscussed above or herein to a subject in need thereof.

In another aspect, the present invention provides a method of treatingan infection, comprising administering any one of the multispecificmolecules discussed above or herein to a subject in need thereof. Insome cases, the infection is a bacterial infection. In some cases, theinfection is a viral infection. In some cases, the infection is a fungalinfection. In some cases, the infection is a parasite infection.

In various embodiments, the target antigen is present at a density offrom 10 to 10,000,000 copies per target cell. In various embodiments,the target antigen is present at a density of from 100 to 10,000,000copies per target cell. In various embodiments, the target antigen ispresent at a density of from 100 to 1,000,000 copies per target cell. Insome embodiments, the target antigen is present at a density of from 50to 10,000. In some embodiments, the target antigen is present at adensity of from 100 to 5000. In some embodiments, the target antigen ispresent at a density of from 100 to 20,000. In some embodiments, thetarget antigen is present at a density of from 500 to 1,000,000 copiesper target cell. In some embodiments, the target antigen is present at adensity of from 1000 to 20,000 copies per target cell. In someembodiments, the target antigen is present at a density of greater than20,000 copies per target cell. In various embodiments, the targetantigen is present at a density of about 10, about 50, about 100, about200, about 300, about 400, about 500, about 1000, about 2000, about3000, about 4000, about 5000, about 6000, about 7000, about 8000, about9000, about 10,000, about 15,000, about 20,000, about 25,000, about50,000, about 75,000, about 100,000, about 200,000, about 300,000, about400,000, about 500,000, about 600,000, about 700,000, about 800,000,about 900,000, about 1,000,000, about 2,000,000, about 3,000,000, about4,000,000 or about 5,000,000 copies per target cell. As used herein, a“low density antigen” is an antigen where no more than 5000 copies ofthe antigen are found on a target cell. References to a low densityantigen include cases in which a cell has no more than 4000, no morethan 3000, no more than 2000, no more than 1000, no more than 900, nomore than 800, no more than 700, no more than 600, no more than 500, nomore than 400, no more than 300, no more than 200, no more than 100, orno more than 50 copies of the target antigen.

In various embodiments, the multispecific molecule is administered incombination with a second therapeutic agent to treat a disease ordisorder. In some cases, the second therapeutic agent comprises abispecific antigen-binding molecule comprising a first antigen-bindingdomain that binds a target antigen (TA) and a second antigen-bindingdomain that binds a T-cell antigen. In some cases, the target antigen isa tumor-cell antigen. In some embodiments, the second therapeutic agentcomprises a bispecific anti-TA x anti-CD28 antibody. In someembodiments, the second therapeutic agent comprises a bispecificanti-EGFR x anti-CD28 antibody. In some embodiments, the secondtherapeutic agent comprises an antibody that binds a check-pointinhibitor on a T cell. In some embodiments, the second therapeutic agentcomprises an anti-PD-1 antibody. In some cases, the multispecificmolecule is administered in combination with two or more secondtherapeutic agents.

In another aspect, the present invention provides for use of any one ofthe multispecific molecules discussed above or herein in the manufactureof a medicament for treating a disease or disorder (e.g., a cancer, oran infection) in a subject in need thereof.

In another aspect, the present invention provides for use of any one ofthe multispecific molecules discussed above or herein in medicine, or totreat a disease or disorder (e.g., a cancer, or an infection).

In another aspect, the present invention provides a multispecificmolecule, as discussed above or herein, for use in medicine, or to treata disease or disorder (e.g., a cancer, or an infection).

In any of the embodiments discussed above or herein, the target antigenmay be a peptide in the context of the groove of a majorhistocompatibility complex (MHC) protein.

In various embodiments, any of the features or components of embodimentsdiscussed above or herein may be combined, and such combinations areencompassed within the scope of the present disclosure. Any specificvalue discussed above or herein may be combined with another relatedvalue discussed above or herein to recite a range with the valuesrepresenting the upper and lower ends of the range, and such ranges areencompassed within the scope of the present disclosure.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate known bispecific antibody and antigen-bindingmolecule formats.

FIGS. 1C, 1E, 1F, 1G, 1H, 1I, 1J, 1K, 1L, 1M, 1N, 1O, 1P, 1Q, 1R and 1Sillustrate bispecific or multispecific antigen-binding molecule formatsin accordance with embodiments of the present invention. In each ofthese formats, a first polypeptide chain comprises both an N-terminaland a C-terminal antigen-binding domain (e.g., a Fab or scFv) thatspecifically binds a T-cell antigen (TCA) (e.g., CD3), and a secondpolypeptide chain comprising at least one antigen-binding domain (e.g.,a Fab or scFv) that binds a target antigen (TA) (e.g., a tumor cellantigen). FIG. 1D illustrates a format in which the two antigen-bindingdomains that specifically bind a T-cell antigen (e.g., CD3) are locatedon different polypeptide chains (at the N-terminus on one polypeptidechain, and at the C-terminus on the second polypeptide chain).

FIG. 2 shows T cell activation induced by molecules having each of theformats illustrated in FIGS. 1A, 1B and 1C compared to a T cell-onlycontrol (ZERO) and a positive control. None of the molecules activated Tcells in the absence of target cells.

FIG. 3 shows the cytotoxic activity of molecules having each of theformats illustrated in FIGS. 1A, 1B and 1C, in the presence of humanPBMC and target cells (A375), compared to a positive control thatinduces maximal cell killing. The CD3-binding domains of the moleculescomprise the variable regions of a 7221G anti-CD3 antibody. The moleculehaving the structure of FIG. 1C was significantly more potent than themolecules having the structures of FIGS. 1A and 1B.

FIGS. 4A, 4B and 4C show the cytotoxic activity of molecules having eachof the formats illustrated in FIGS. 1A, 1B and 1C, in the presence ofhuman PBMC and target cells (A375), in combination with an anti-PD-1antibody (FIG. 4A), a co-stimulatory bispecific EGFR x CD28 antibody(FIG. 4B), or both an anti-PD-1 antibody and a co-stimulatory bispecificEGFR x CD28 antibody (FIG. 4C) compared to a positive control thatinduces maximal cell killing. The CD3-binding domains of the moleculescomprise the variable regions of a 7221G anti-CD3 antibody. The moleculehaving the structure of FIG. 1C was significantly more potent incombination with these additional antibodies than the molecules havingthe structures of FIGS. 1A and 1B.

FIG. 5 shows the measured cytokine levels of the molecule having thestructure of FIG. 1C (right panel) compared to the molecule having thestructure of FIG. 1A (left panel) at the point of maximal antibodyconcentration shown in FIGS. 4A, 4B and 4C. The CD3-binding domains ofthe molecules comprise the variable regions of a 7221G anti-CD3antibody. The molecule having the structure of FIG. 1C does not showgreater levels of cytokine release in spite of the significantly greatercytotoxic activity.

FIGS. 6A, 6B, 6C and 6D show binding of the molecule having thestructure of FIG. 1C and modified versions of this molecule (withinactive domains—noted by an X in the legend) to Raji cells (FIG. 6A) orA375 cells (FIG. 6C) overexpressing a MAGEA4 peptide, or CD3+ Jurkatcells (FIGS. 6B and 6D). The CD3-binding domains of the moleculesillustrated in FIGS. 6A and 6B comprise the variable regions of a 7195Panti-CD3 antibody. The CD3-binding domains of the molecules illustratedin FIGS. 6C and 6D comprise the variable regions of a 7221G anti-CD3antibody. As shown in FIGS. 6A, 6B, 6C and 6D, the presence of twoactive antigen-binding domains improved binding to the target antigens,and similar binding was observed irrespective of the source of theanti-CD3 binding domains. As illustrated in these figures, binding wasmost affected when the N-terminal Fab domain was removed.

FIGS. 7A and 7B show the cytotoxic activity of the same molecules shownin FIGS. 6A and 6B (FIG. 7A), and FIGS. 6C and 6D (FIG. 7B). Themolecule having the structure of FIG. 1C showed the greatest cytotoxicpotency, followed by the molecules with two active T-cell antigen (e.g.,CD3) binding domains. A similar pattern of cytotoxicity was observedirrespective of the source of the anti-CD3 binding domains.

FIGS. 8A and 8B show binding of the molecule having the structure ofFIG. 1C and modified versions of this molecule (with C-terminal Fabdomains or inactive domains—noted by an X in the legend) to Raji cellsoverexpressing a MAGEA4 peptide (FIG. 8A) or CD3+ Jurkat cells (FIG.8B). The CD3-binding domains of the molecules comprise the variableregions of a 7195P anti-CD3 antibody. As shown in FIGS. 8A and 8B,C-terminal scFv domains provided superior binding to the target antigenscompared to C-terminal Fab domains.

FIG. 9 shows the cytotoxic activity of the same molecules shown in FIGS.8A and 8B. The CD3-binding domains of the molecules comprise thevariable regions of a 7195P anti-CD3 antibody. The molecule having thestructure of FIG. 1C showed the greatest cytotoxic potency, followed bythe molecule having the structure of FIG. 1E.

FIGS. 10A and 10B show binding of the molecules having the structures ofFIGS. 1C and 1D to A375 cells overexpressing a MAGEA4 peptide (FIG.10A), or CD3+ Jurkat cells (FIG. 10B). The CD3-binding domains of themolecules comprise the variable regions of a 7221G anti-CD3 antibody.The two molecules showed similar binding to both cell types relative toone another.

FIGS. 11A and 11B show the cytotoxic activity of the same moleculesshown in FIGS. 10A and 10B on A375 cells from two different donorsources. The CD3-binding domains of the molecules comprise the variableregions of a 7221G anti-CD3 antibody. The molecule having the structureof FIG. 1C was more potent than the molecule having the structure ofFIG. 1D.

FIGS. 12A and 12B show the relative cytotoxic activity and potency ofmolecules having the structures of FIG. 1C and FIG. 1F, respectively, ascompared to a molecule having the structure of FIG. 1A. The moleculeswere tested individually, and in combination with a co-stimulatorybispecific EGFR x CD28 antibody and an anti-PD-1 antibody, as discussedin Example 7. The CD3-binding domains of the molecules comprise thevariable regions of a 7195P anti-CD3 antibody. The molecule having thestructure of FIG. 1F targets two different epitopes of the same targetantigen with the two TA antigen-binding domains, whereas the moleculehaving the structure of FIG. 1C targets the same epitope of the targetantigen with the two TA antigen-binding domains. The molecule having thestructure of FIG. 1F was more potent than the molecule having thestructure of FIG. 1C, and both molecules were more potent that themolecule having the structure of FIG. 1A. In each case, the combinationof these molecules with the co-stimulatory bispecific antibody and theanti-PD-1 antibody produced even greater cytotoxic potency, similar tothe results shown in FIGS. 4A-4C.

FIG. 13 shows the relative binding affinity for molecules having thestructure of FIG. 1F, in which the CD3-binding domains are derived fromanti-CD3 antibodies with strong, moderate, or weak binding affinity toCD3. The “strong” binding domains are derived from the 7195P anti-CD3antibody. The “moderate” binding domains are derived from the 7221Ganti-CD3 antibody. The “weak” binding domains are derived from the7221G20 anti-CD3 antibody. The references to, e.g., “strong/strong”refer, respectively, to the Fab anti-CD3 binding domain and the scFcanti-CD3 binding domain. As expected, binding to CD3-positive Jurkatcells correlates with the strength of the affinity of the anti-CD3binding domains in the molecules.

FIGS. 14A and 14B show the relative cytotoxic activity and potency ofthe molecules shown in FIG. 13 in MAGEA4-positive A375 cells. Themolecules were tested individually (FIG. 14A), and in combination with aco-stimulatory bispecific EGFR x CD28 and an anti-PD-1 antibody (FIG.14B), as discussed in Example 8. There is a clear correlation betweenthe strength of the anti-CD3 binding domains and the potency of themolecules. The “Control” is a positive control that targets the scaffoldof all HLA molecules to provide a maximum cytotoxicity against which tocompare the other molecules.

FIGS. 15A and 15B show the relative cytotoxic activity and potency ofthe molecules shown in FIG. 13 in MAGEA4-positive ScaBER cells. Themolecules were tested individually (FIG. 15A), and in combination with aco-stimulatory bispecific EGFR x CD28 and an anti-PD-1 antibody (FIG.15B), as discussed in Example 8. There is a clear correlation betweenthe strength of the anti-CD3 binding domains and the potency of themolecules. The “Control” is a positive control that targets the scaffoldof all HLA molecules to provide a maximum cytotoxicity against which tocompare the other molecules.

FIGS. 16A, 16B and 16C show the relative binding affinity for moleculeshaving the structures of FIGS. 1A (Molecule C), 1C (Molecule B), and IF(Molecules A and D) to NYESO-1-positive cells (FIG. 16A), MAGEA4(peptide 1)-positive cells (FIG. 16B) and MAGEA4 (peptide 2)-positivecells (FIG. 16C). As expected, Molecule D, without an NYESO-1 bindingdomain does not bind to the NYESO-1 expressing cells (FIG. 16A), and themolecules that lack the relevant MAGEA4 binding domain do not bind tothe MAGEA4-expressing cells, as shown in FIGS. 16B and 16C. TheCD3-binding domains of the molecules comprise the variable regions of a7195P anti-CD3 antibody. The “HLA Targeting Bispecific” positive controlbinds HLA molecules and CD3. The “Isotype Control Multispecific” is amolecule having the structure of FIG. 1C with binding domains to anirrelevant target antigen.

FIGS. 17A and 17B show the relative cytotoxic activity and potency ofmolecules having the structures of FIG. 1C and FIG. 1F, respectively, ascompared to a positive control having the structure of FIG. 1A, whichbinds HLA molecules and CD3. The isotype controls included a moleculewith the structure of FIG. 1C with binding domains to an irrelevanttarget antigen, and a molecule with the structure of FIG. 1A withbinding domains to CD3 and an irrelevant target antigen. The moleculeswere tested individually, and in combination with a co-stimulatorybispecific EGFR x CD28 antibody and an anti-PD-1 antibody, as discussedin Example 9. The CD3-binding domains of the molecules comprise thevariable regions of a 7195P anti-CD3 antibody. The molecule having thestructure of FIG. 1F targets two different antigens (NYESO-1 and MAGEA4)with the two TA antigen-binding domains, whereas the molecule having thestructure of FIG. 1C targets a single antigen with both of the two TAantigen-binding domains. The molecule having the structure of FIG. 1Fand targeting two different antigens was more potent than the moleculehaving the structure of FIG. 1C. In each case, the combination of thesemolecules with the co-stimulatory bispecific antibody and the anti-PD-1antibody produced even greater cytotoxic potency, relative to themolecule alone, similar to the results shown in FIGS. 4A-4C.

FIGS. 17C and 17D shown the relative T-cell activation of the moleculesdiscussed in connection with FIGS. 17A and 17B.

FIGS. 18A and 18B show the relative cytotoxic activity and potency ofmolecules having the structures of FIG. 1C and FIG. 1F, respectively, ascompared to a molecule having the structure of FIG. 1A. The positivecontrol is a molecule with the structure of FIG. 1A that binds humanleukocyte antigen (HLA) molecules and CD3. The isotype controls includeda molecule with the structure of FIG. 1C with binding domains to anirrelevant target antigen, and a molecule with the structure of FIG. 1Awith binding domains to CD3 and an irrelevant target antigen. Themolecules were tested individually, and in combination with aco-stimulatory bispecific EGFR x CD28 antibody and an anti-PD-1antibody, as discussed in Example 9. The CD3-binding domains of themolecules comprise the variable regions of a 7195P anti-CD3 antibody. Asshown in FIG. 18A, the molecule having the structure of FIG. 1F(targeting two distinct epitopes of MAGEA4) is more potent than themolecule having the structure of FIG. 1C (targeting a single epitopewith both TA-binding domains), and both molecules are more potent thanthe molecule having the structure of FIG. 1A. Similarly, as shown inFIG. 18B, the molecule having the structure of FIG. 1F (targeting twodifferent antigens) is more potent than the molecule having thestructure of FIG. 1C (targeting a single antigen with both TA-bindingdomains), and both molecules are more potent than the molecule havingthe structure of FIG. 1A. In each case, the combination of thesemolecules with the co-stimulatory bispecific antibody and the anti-PD-1antibody produced even greater cytotoxic potency, relative to themolecule alone, similar to the results shown in FIGS. 4A-4C.

FIGS. 18C, 18D, 18E and 18F show the relative T-cell activation of themolecules discussed in connection with FIGS. 18A and 18B.

FIGS. 19A and 19B show the cytotoxic activity and potency, and T-cellactivation, respectively, of a molecule having the structure of FIG. 1Frelative to a combination of two molecules having the structure of FIG.1A, in which the combination of the two molecules binds the same pair oftarget antigens as the molecule having the structure of FIG. 1F. Asshown in FIGS. 19A and 19B, the molecule having the structure of FIG. 1Fmore potently kills the tumor cells and increases T-cell activation thandoes the combination of the two molecules having the structure of FIG.1A.

DETAILED DESCRIPTION

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to particular methods andexperimental conditions described, as such methods and conditions mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The term “T cell” refers to immune cells expressing CD3, including CD4+cells (helper T cells), CD8+ cells (cytotoxic T cells), regulatory Tcells (Tregs), and tumor infiltrating lymphocytes.

The expression “T-cell antigen” refers to a cell-surface expressedprotein present on a T cell, and includes “co-stimulatory molecules.” A“co-stimulatory molecule” refers to a protein expressed by a T cell thatbinds a cognate ligand or receptor (e.g., on an antigen-presenting cell)to provide a stimulatory signal, which, in combination with the primarysignal provided by engagement of the T cell's TCR with a peptide/MHC,stimulates the activity of the T cell. Stimulation of a T cell caninclude activation, proliferation and/or survival of the T cell.

As used herein, the expression “cell surface-expressed” or “cell-surfacemolecule” means one or more protein(s) that is/are expressed on thesurface of a cell in vitro or in vivo, such that at least a portion ofthe protein is exposed to the extracellular side of the cell membraneand is accessible to an antigen-binding portion of an antibody or anantigen-binding domain of the multispecific antigen-binding moleculesdiscussed herein.

The expression “CD3,” as used herein, refers to an antigen which isexpressed on T cells as part of the multimolecular T cell receptor (TCR)and which consists of a homodimer or heterodimer formed from theassociation of two of four receptor chains: CD3-epsilon, CD3-delta,CD3-zeta, and CD3-gamma. All references to proteins, polypeptides andprotein fragments herein are intended to refer to the human version ofthe respective protein, polypeptide or protein fragment unlessexplicitly specified as being from a non-human species. Thus, theexpression “CD3” means human CD3 unless specified as being from anon-human species, e.g., “mouse CD3,” “monkey CD3,” etc.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). Theantigen-binding domains of the present invention may bind soluble CD3and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

As used herein, the expression “cell surface-expressed CD3” means one ormore CD3 protein(s) that is/are expressed on the surface of a cell invitro or in vivo, such that at least a portion of a CD3 protein isexposed to the extracellular side of the cell membrane and is accessibleto an antigen-binding portion of an antibody. “Cell surface-expressedCD3” includes CD3 proteins contained within the context of a functionalT cell receptor in the membrane of a cell. The expression “cellsurface-expressed CD3” includes CD3 protein expressed as part of ahomodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon,delta/epsilon, and zeta/zeta CD3 dimers). The expression, “cellsurface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon,CD3-delta or CD3-gamma) that is expressed by itself, without other CD3chain types, on the surface of a cell. A “cell surface-expressed CD3”can comprise or consist of a CD3 protein expressed on the surface of acell which normally expresses CD3 protein. Alternatively, “cellsurface-expressed CD3” can comprise or consist of CD3 protein expressedon the surface of a cell that normally does not express human CD3 on itssurface but has been artificially engineered to express CD3 on itssurface.

The term “antigen-binding domain” refers to that portion of amultispecific molecule or a corresponding antibody that bindsspecifically to a predetermined antigen (e.g., CD3 or a tumor associatedantigen). References to a “corresponding antibody” refer to the antibodyfrom which the CDRs or variable regions (HCVR and LCVR) used in amultispecific molecule are derived. For example, the FIG. 1C structuredmolecules discussed in the examples include Fabs and scFvs with variableregions derived from specific anti-CD3 antibodies and anti-MAGEA4antibodies. These antibodies are the “corresponding antibodies” to therespective multispecific molecules.

The term “multispecific antigen-binding molecule” includes moleculesthat bind two or more (e.g., three or four) different epitopes orantigens. In some cases, the multispecific antigen-binding molecules arebispecific. In some cases, the multispecific antigen-binding moleculesare trispecific. In some cases, the multispecific antigen-bindingmolecules are tetraspecific.

The term “antibody” means any antigen-binding molecule or molecularcomplex comprising at least one complementarity determining region (CDR)that specifically binds to or interacts with a particular antigen (e.g.,CD3 or a target antigen (TA)). The term “antibody” includesimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). The term “antibody” alsoincludes immunoglobulin molecules consisting of four polypeptide chains,two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-TA antibody or anti-CD3 antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (Vii) V_(H)-C_(L);V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

In certain embodiments of the invention, the antibodies are humanantibodies. The term “human antibody” is intended to include antibodieshaving variable and constant regions derived from human germlineimmunoglobulin sequences. The human antibodies may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo), for example in the CDRs and in particularCDR3. However, the term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

The antibodies discussed herein may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody” is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

The antibodies referenced herein may be isolated antibodies. An“isolated antibody,” as used herein, means an antibody that has beenidentified and separated and/or recovered from at least one component ofits natural environment. For example, an antibody that has beenseparated or removed from at least one component of an organism, or froma tissue or cell in which the antibody naturally exists or is naturallyproduced, is an “isolated antibody.” An isolated antibody also includesan antibody in situ within a recombinant cell. Isolated antibodies areantibodies that have been subjected to at least one purification orisolation step. An isolated antibody may be substantially free of othercellular material and/or chemicals.

The antibodies referenced herein may comprise one or more amino acidsubstitutions, insertions and/or deletions in the framework and/or CDRregions of the heavy and light chain variable domains as compared to thecorresponding germline sequences from which the antibodies were derived.Such mutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

A “multimerization domain” or “multimerizing domain” is anymacromolecule that has the ability to associate (covalently ornon-covalently) with a second macromolecule of the same or similarstructure or constitution. For example, a multimerization domain may bea polypeptide comprising an immunoglobulin C_(H)3 domain. A non-limitingexample of a multimerization domain is an Fc portion of animmunoglobulin, e.g., an Fc domain of an IgG selected from the isotypesIgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotypegroup. In certain embodiments, the multimerization domain is an Fcfragment or an amino acid sequence of 1 to about 200 amino acids inlength containing at least one cysteine residue. In other embodiments,the multimerization domain is a cysteine residue or a shortcysteine-containing peptide. Other multimerization domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif. In some embodiments, themultimerizing domain is an immunoglobulin Fc domain and themultispecific antigen-binding molecules of the present invention areformed by association of two such Fc domains via interchain disulfidebonding as in a conventional antibody.

The terms “nucleic acid” or “polynucleotide” refers to nucleotidesand/or polynucleotides, such as deoxyribonucleic acid (DNA) orribonucleic acid (RNA), oligonucleotides, fragments generated by thepolymerase chain reaction (PCR), and fragments generated by any ofligation, scission, endonuclease action, and exonuclease action. Nucleicacid molecules can be composed of monomers that are naturally-occurringnucleotides (such as DNA and RNA), or analogs of naturally-occurringnucleotides (e.g., enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Modified nucleotides can havealterations in sugar moieties and/or in pyrimidine or purine basemoieties. Sugar modifications include, for example, replacement of oneor more hydroxyl groups with halogens, alkyl groups, amines, and azidogroups, or sugars can be functionalized as ethers or esters. Moreover,the entire sugar moiety can be replaced with sterically andelectronically similar structures, such as aza-sugars and carbocyclicsugar analogs. Examples of modifications in a base moiety includealkylated purines and pyrimidines, acylated purines or pyrimidines, orother well-known heterocyclic substitutes. Nucleic acid monomers can belinked by phosphodiester bonds or analogs of such linkages. Nucleicacids can be either single stranded or double stranded.

The term “recombinant,” as used herein, is intended to include allmolecules that are prepared, expressed, created or isolated byrecombinant means, such as multispecific molecules (e.g. bispecificmolecules) expressed using a recombinant expression vector transfectedinto a host cell, multispecific molecules (e.g., bispecific molecules)isolated from an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or multispecific molecules prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin and/or MHC gene sequences to other DNA sequences. Suchrecombinant multispecific molecules can include antigen-binding domainshaving variable and constant regions derived from human germlineimmunoglobulin sequences.

The term “subject” or “patient” as used herein includes all members ofthe animal kingdom including non-human primates and humans. In oneembodiment, patients are humans with a disease or disorder, e.g., aninfection or a cancer.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

The terms “vector” and “expression vector” include, but are not limitedto, a viral vector, a plasmid, an RNA vector or a linear or circular DNAor RNA molecule which may consist of chromosomal, non-chromosomal,semi-synthetic or synthetic nucleic acids. In some cases, the vectorsare those capable of autonomous replication (episomal vector) and/orexpression of nucleic acids to which they are linked (expressionvectors). Large numbers of suitable vectors are known to those of skillin the art and are commercially available. Viral vectors includeretrovirus, adenovirus, parvovirus (e.g., adenoassociated viruses),coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g.,influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitisvirus), paramyxovirus (e.g. measles and Sendai), positive strand RNAviruses such as picornavirus and alphavirus, and double-stranded DNAviruses including adenovirus, herpesvirus (e.g., Herpes Simplex virustypes 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g.,vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus,togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, andhepatitis virus, for example. Examples of retroviruses include: avianleukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses,HTLV-BLV group, and lentivirus.

Multispecific Antigen-Binding Molecules

The multispecific antigen-binding molecules (e.g., bispecific ortrispecific or tetraspecific) of the present invention comprise (a) afirst polypeptide comprising, from N-terminus to C-terminus (i) a firstantigen-binding domain that specifically binds a T cell antigen, (ii) afirst multimerizing domain, and (iii) a second antigen-binding domainthat specifically binds a T cell antigen; and (b) a second polypeptidecomprising, from N-terminus to C-terminus (i) a third antigen-bindingdomain that specifically binds a target antigen, and (ii) a secondmultimerizing domain, wherein the first and the second multimerizingdomains associate with one another (e.g., via interchain disulfidebonding) to form the molecule.

In some embodiments, the multispecific antigen-binding molecules (e.g.,bispecific or trispecific or tetraspecific) of the present inventioncomprise (a) a first polypeptide comprising, from N-terminus toC-terminus (i) a first antigen-binding domain that specifically binds aT cell antigen, (ii) a first multimerizing domain, and (iii) a secondantigen-binding domain that specifically binds a T cell antigen; and (b)a second polypeptide comprising, from N-terminus to C-terminus (i) athird antigen-binding domain that specifically binds a target antigen,(ii) a second multimerizing domain, and (iii) a fourth antigen-bindingdomain that specifically binds a target antigen, wherein the first andthe second multimerizing domains associate with one another (e.g., viainterchain disulfide bonding) to form the molecule.

The antigen-binding domains referenced above and herein can be Fabdomains, comprising a heavy chain variable region (HCVR) and a heavychain CH1 domain paired with a light chain variable region (LCVR) and aCL domain. The antigen-binding domains referenced above and herein canalso be single chain variable fragment (scFv) domains, comprising a HCVRand LCVR connected together by a short peptide linker of, e.g., fromabout 10 to about 25 amino acids. Specific linkers include (G4S)_(n)linkers, wherein n=1-10, or n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Insome cases, the linker between the HCVR and LCVR of each scFv is (G45)₄.Unless otherwise defined, the antigen-binding domains of themultispecific molecules of the present invention can be all Fab domains,all scFv domains, or a combination of Fab domains and scFv domains. Insome cases, one or more of the antigen-binding domains is a Fab domain.In some cases, one or more of the antigen-binding domains is a scFvdomain. In some cases, the first antigen-binding domain and the thirdantigen-binding domain are Fab domains. In some cases, the secondantigen-binding domain is an scFv domain. In some cases, the fourthantigen-binding domain is an scFv domain. In some cases, the first andthird antigen-binding domains are Fab domains, and the second and fourthantigen-binding domains are scFv domains. In some cases, the first,second and third antigen-binding domains are Fab domains. In some cases,the first, second, third and fourth antigen-binding domains are Fabdomains.

In various embodiments, the scFv domains are connected to the C-terminusof the respective multimerizing domain via a linker peptide. In somecases, the linker is between 1-10 amino acids long. In some embodiments,the linker is between 1-20 amino acids long. In this regard, the linkermay be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 amino acids long. In some embodiments, the linker may be 21, 22,23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. Ranges including thenumbers discussed herein are also encompassed within this disclosure,e.g., a linker 10-30 amino acids long. In some embodiments, the linkersare flexible linkers. Suitable linkers can be readily selected and canbe of any of a suitable of different lengths, such as from 1 amino acid(e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids,from 3 amino acids to 12 amino acids, including 4 amino acids to 10amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 aminoacids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6,or 7 amino acids. Exemplary flexible linkers include glycine polymers(G)n, glycine-serine polymers (GS)_(n), where n is an integer of atleast one (e.g., from 1-20), glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers known in the art. Specific linkersinclude (G4S)_(n) linkers, wherein n=1-10, or n is 1, 2, 3, 4, 5, 6, 7,8, 9, or 10. In some cases, the linker between each scFv domain and theC-terminus of the respective multimerizing domain is (G45)₃.

In those embodiments in which one or more antigen-binding domains is anscFv, the scFv can be a stabilized scFv, in which one or moremodifications is made to the HCVR and/or LCVR sequence in order toproduce and maintain a proper conformation of the scFv. In someembodiments, the scFv includes cysteine mutations at residue 44 of theHCVR and residue 100 of the LCVR (Kabat numbering) to produceinter-disulfide bonding between the variable regions (see, Zhao et al.,Int. J. Mol. Sci, 12:1-11, 2011; and Weatherill et al., ProteinEngineering, Design and Selection, 25 (7):321-329, 2012). In someembodiments, the scFv includes mutations at residue 39 of the HCVR andresidue 38 of the LCVR (Kabat numbering) to modify the glutamineresidues to glutamic acid or lysine residues to inhibit conformationalisomerization (see, Igawa et al., Protein Engineering, Design andSelection, 23 (8):667-677, 2010).

In various embodiments, the LCVR (and optionally the CL) of any of theantigen-binding domains can be a cognate LCVR that corresponds to theHCVR, or the LCVR can be a universal LCVR (and optionally CL) common tomultiple antigen-binding domains. In some embodiments, the light chainof the Fab domains is a common light chain. In some embodiments, thelight chain of the Fab domains is a cognate light chain corresponding tothe target antigen binding domain, and the light chain is common to bothFab domains. In some embodiments, the LCVR of the scFv domains is acognate LCVR. In some embodiments, the light chain of the Fab domains isa common light chain and the LCVR of the scFv domains is a cognate LCVR.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a second scFv that specifically binds a targetantigen, wherein the first and the second immunoglobulin domainsassociate with one another via disulfide bonding to form the molecule.An exemplary structure for such a molecule is illustrated in FIG. 1C.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a secondFab that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a third Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a fourth Fab that specifically binds a targetantigen, wherein the first and the second immunoglobulin domainsassociate with one another via disulfide bonding to form the molecule.An exemplary structure for such a molecule is illustrated in FIG. 1E.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a first target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a second scFv that specificallybinds a second target antigen different from the first target antigen,wherein the first and the second immunoglobulin domains associate withone another via disulfide bonding to form the molecule. An exemplarystructure for such a molecule is illustrated in FIG. 1F.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a secondFab that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a third Fabthat specifically binds a first target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a fourth Fab that specifically bindsa second target antigen different from the first target antigen, whereinthe first and the second immunoglobulin domains associate with oneanother via disulfide bonding to form the molecule. An exemplarystructure for such a molecule is illustrated in FIG. 1G.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a second scFv that specifically binds a T cellantigen, wherein the first and the second immunoglobulin domainsassociate with one another via disulfide bonding to form the molecule.An exemplary structure for such a molecule is illustrated in FIG. 1H.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a secondFab that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a third Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a fourth Fab that specifically binds a T cellantigen, wherein the first and the second immunoglobulin domainsassociate with one another via disulfide bonding to form the molecule.An exemplary structure for such a molecule is illustrated in FIG. 1I.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a secondFab that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a target antigen, and (ii) a secondimmunoglobulin Fc domain, wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule. An exemplary structure for such a molecule isillustrated in FIG. 1J.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a Tcell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a secondFab that specifically binds a T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a third Fabthat specifically binds a target antigen, and (ii) a secondimmunoglobulin Fc domain, wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule. An exemplary structure for such a molecule isillustrated in FIG. 1K.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a second T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a second scFv that specifically binds a targetantigen, wherein the first and the second immunoglobulin domainsassociate with one another via disulfide bonding to form the molecule.An exemplary structure for such a molecule is illustrated in FIG. 1L.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) asecond Fab that specifically binds a second T cell antigen; and (b) asecond polypeptide comprising, from N-terminus to C-terminus (i) a thirdFab that specifically binds a target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a fourth Fab that specifically bindsa target antigen, wherein the first and the second immunoglobulindomains associate with one another via disulfide bonding to form themolecule. An exemplary structure for such a molecule is illustrated inFIG. 1M.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a second T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a first target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a second scFv that specificallybinds a second target antigen different from the first target antigen,wherein the first and the second immunoglobulin domains associate withone another via disulfide bonding to form the molecule. An exemplarystructure for such a molecule is illustrated in FIG. 1N.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) asecond Fab that specifically binds a second T cell antigen; and (b) asecond polypeptide comprising, from N-terminus to C-terminus (i) a thirdFab that specifically binds a first target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a fourth Fab that specifically bindsa second target antigen different from the first target antigen, whereinthe first and the second immunoglobulin domains associate with oneanother via disulfide bonding to form the molecule. An exemplarystructure for such a molecule is illustrated in FIG. 1O.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) a firstscFv that specifically binds a second T cell antigen; and (b) a secondpolypeptide comprising, from N-terminus to C-terminus (i) a second Fabthat specifically binds a target antigen, (ii) a second immunoglobulinFc domain, and (iii) a second scFv that specifically binds a T cellantigen (optionally may bind the first T cell antigen, the second T cellantigen, or a third T cell antigen), wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule. An exemplary structure for such a molecule isillustrated in FIG. 1P.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) asecond Fab that specifically binds a second T cell antigen; and (b) asecond polypeptide comprising, from N-terminus to C-terminus (i) a thirdFab that specifically binds a target antigen, (ii) a secondimmunoglobulin Fc domain, and (iii) a fourth Fab that specifically bindsa T cell antigen (optionally may bind the first T cell antigen, thesecond T cell antigen, or a third T cell antigen), wherein the first andthe second immunoglobulin domains associate with one another viadisulfide bonding to form the molecule. An exemplary structure for sucha molecule is illustrated in FIG. 1Q.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) asecond Fab that specifically binds a second T cell antigen; and (b) asecond polypeptide comprising, from N-terminus to C-terminus (i) asecond Fab that specifically binds a target antigen, and (ii) a secondimmunoglobulin Fc domain, wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule. An exemplary structure for such a molecule isillustrated in FIG. 1R.

In some embodiments, the multispecific antigen-binding molecules of thepresent invention comprise: (a) a first polypeptide comprising, fromN-terminus to C-terminus (i) a first Fab that specifically binds a firstT cell antigen, (ii) a first immunoglobulin Fc domain, and (iii) asecond Fab that specifically binds a second T cell antigen; and (b) asecond polypeptide comprising, from N-terminus to C-terminus (i) a thirdFab that specifically binds a target antigen, and (ii) a secondimmunoglobulin Fc domain, wherein the first and the secondimmunoglobulin domains associate with one another via disulfide bondingto form the molecule. An exemplary structure for such a molecule isillustrated in FIG. 1S.

Unless otherwise defined, and when present, the fourth antigen-bindingdomain can specifically bind a target antigen or a T cell antigen. Insome cases, the third antigen-binding domain and the fourthantigen-binding domain specifically bind distinct target antigens(different epitopes on the same protein, or different proteins). In somecases, the distinct target antigens are expressed on the surface of thesame target cell (e.g., tumor cell). In some cases, the thirdantigen-binding domain and the fourth antigen-binding domainspecifically bind the same target antigen (the same epitope on the sameprotein). In various embodiments, the first and second antigen-bindingdomains, and the fourth antigen-binding domain (when present, anddirected to a T-cell antigen) can bind the same or distinct T-cellantigens, as illustrated in the figures. In some cases, the first,second and fourth antigen-binding domains specifically bind distinctT-cell antigens (different epitopes on the same protein, or differentproteins). In some cases, the first, second and fourth antigen-bindingdomains specifically bind the same T-cell antigen (the same epitope onthe same protein). In some cases, the distinct T-cell antigens are aco-stimulatory molecule (e.g., CD28) and a check-point inhibitor (e.g.,PD-1) on the surface of a T cell. In such embodiments, the multispecificmolecules of the invention can provide a costimulatory signal to the Tcell as well as prevent checkpoint inhibition. As used herein, referenceto “same” target antigen or T-cell antigen does not necessarily meanthat the antigen-binding domains are binding to the same surfacemolecule, but rather that the antigen-binding domains have the samespecificity (e.g., they each bind CD3 or a TA). Similarly, references toa “distinct” target antigen or T-cell antigen mean that it is differentfrom another target antigen (e.g., MAGEA4 vs. EGFR) or another T-cellantigen (e.g., CD28 vs. PD-1), or is another epitope on the sameprotein.

In any of the embodiments discussed above or herein, the target antigencan be a tumor-associated antigen or an infection-associated antigen(e.g., a viral antigen, a bacterial antigen, a fungal antigen, or anantigen expressed by a parasite). In some cases, the target antigen is atumor-associated antigen. In some cases, the target antigen is aninfection-associated antigen. In some cases, the target antigen is aviral antigen. In some cases, the target antigen is a bacterial antigen.In some cases, the target antigen is a fungal antigen. In some cases,the target antigen is an antigen expressed by a parasite.

In some cases, the target antigen is a peptide in the context of thegroove (PiG) of a major histocompatibility complex (MHC) protein. Insome embodiments, the PiG is a peptide consisting of about 5 to about 40amino acid residues, from about 6 to about 30 amino acid residues, fromabout 8 to about 20 amino acid residues, or about 9, 10, or 11 aminoacid residues. In some cases, the PiG is a fragment of atumor-associated antigen, a viral antigen, a bacterial antigen, a fungalantigen, or a parasite antigen. In various embodiments, the targetantigen is a peptide in the context of the groove of any class, subtypeor allele of human leukocyte antigen, including any of HLA-A, HLA-B,HLA-C, HLA-DR, HLA-DQ or HLA-DP. In some embodiments, the target antigenis a peptide/MHC complex. In some cases, the peptide in the peptide/MHCcomplex is a fragment of a tumor-associated antigen, a fragment of abacterial antigen, a fragment of a viral antigen, a fragment of a fungalantigen, or a fragment of a parasite antigen.

In some cases, the antigen is a tumor-associated antigen or an antigenexpressed by a tumor cell. In some embodiments, the tumor-associatedantigen is selected from the group consisting of AFP, ALK, BAGEproteins, BIRC5 (survivin), BIRC7, β-catenin, brc-abl, BRCA1, BORIS,CA9, carbonic anhydrase IX, caspase-8, CALR, CCR5, CD19, CD20 (MS4A1),CD22, CD40, CD70, CDK4, CEA, cyclin-B1, CYP1B1, EGFR, EGFRvIII,ErbB2/Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGEproteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100,Her2, HLA/B-raf, HLA/k-ras, HLA/MAGE-A3, hTERT, IL-10, LMP2, MAGEproteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin,ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17,NY-BR1, NY-BR62, NY-BR85, NY-ESO1, p15, p53, PAP, PAX3, PAX5, PCTA-1,PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1,SART-3, STEAP1, STEAP2, TAG-72, TGF-β, TMPRSS2, Thompson-nouvelleantigen (Tn), TRP-1, TRP-2, tyrosinase, and uroplakin-3.

In some cases, the antigen is a viral antigen or a bacterial antigen. Insome embodiments, the viral antigen is associated with or expressed by avirus selected from the group consisting of adenovirus, astrovirus,chikungunya, cytomegalo, dengue, ebola, EBV, hantavirus, HBsAg,hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes,HIV, HPIV, HTLV, influenza, Japanese encephalitis virus, lassa, measles,metapneumovirus, mumps, norovirus, oropauche, HPV, parvovirus,rotavirus, RSV, rubella, SARS, TBEV, usutu, vaccina, varicella, WestNile, yellow fever, and zika, or the bacterial antigen is derived from abacterium selected from the group consisting of methicillin-resistantStaphylococcus aureus (MRSA), Clostridium difficile,carbapenum-resistant Enterobacteriaceae, drug-resistant Neisseriagonorrhoeae, multidrug-resistant Acinetobacter, drug-resistantCampylobacter, Fluconazole-resistant Candida, extended-spectrumβ-lactamase producing bacteria, Vancomycin-resistant Enterococcus,multidrug-resistant Pseudomonas aeruginosa, drug-resistant non-typhoidalSalmonella, drug-resistant Salmonella serotype typhi, drug-resistantShigella, drug-resistant Streptococcus pneumoniae, drug-resistanttuberculosis, Vancomycin-resistant Staphylococcus aureus,Erythomycin-resistant group A Streptococcus, and Clindamycin-resistantgroup B Streptococcus.

In any of the embodiments discussed above or herein, the T cell antigencan be an antigen expressed at the surface of a T cell, a T cellreceptor complex antigen, a co-stimulatory molecule or a check pointinhibitor on a T cell, CD3, CD27, CD28, 4-1BB or PD-1. In some cases,the T cell antigen is a T cell receptor complex antigen. In some cases,the T cell antigen is CD3. In some cases, the T cell antigen is aco-stimulatory molecule or a check-point inhibitor on a T cell. In somecases, the T cell antigen is selected from the group consisting of CD27,CD28, 4-1BB and PD-1. In some cases, the T cell antigen is selected fromthe group consisting of CD3, CD27, CD28, 4-1BB and PD-1. In some cases,the T cell antigen is selected from the group consisting of CD28, ICOS,HVEM, CD27, 4-1BB, 0X40, DR3, GITR, CD30, SLAM, CD2, 2B4, CD226, TIM1,and TIM2.

In certain embodiments in which the T cell antigen is CD3, theCD3-binding domain binds to human CD3 and induces human T cellactivation. In certain embodiments, the CD3-binding domain binds weaklyto human CD3 and induces human T cell activation. In some embodiments,the CD3-binding domain binds weakly to human CD3 and inducestumor-associated antigen-expressing cell killing. In some embodiments,the CD3-binding domain binds or associates weakly with human andcynomolgus (monkey) CD3, yet the binding interaction is not detectableby in vitro assays known in the art. In some embodiments, theCD3-binding domain binds with weak affinity to human CD3. In someembodiments, the CD3-binding domain binds with moderate affinity tohuman CD3. In some embodiments, the CD3-binding domain binds with highaffinity to human CD3. In some embodiments, the CD3-binding domain bindsto human CD3 (e.g., at 25° C.) with a K_(D) of less than about 15 nM asmeasured by surface plasmon resonance (e.g., mAb-capture orantigen-capture format) or a substantially similar assay. In someembodiments, the CD3-binding domain binds human CD3 with an K_(D) valueof greater than about 15 nM, greater than about 20 nM, greater thanabout 30 nM, greater than about 40 nM, greater than about 50 nM, greaterthan about 60 nM, greater than about 100 nM, greater than about 200 nM,or greater than about 300 nM, as measured in a surface plasmon resonancebinding assay (e.g., mAb-capture or antigen-capture format) or asubstantially similar assay. In some embodiments, the antibodies orantigen-binding fragments of the present invention bind CD3 with a K_(D)of less than about 5 nM, less than about 2 nM, less than about 1 nM,less than about 800 pM, less than about 600 pM, less than about 500 pM,less than about 400 pM, less than about 300 pM, less than about 200 pM,less than about 180 pM, less than about 160 pM, less than about 140 pM,less than about 120 pM, less than about 100 pM, less than about 80 pM,less than about 60 pM, less than about 40 pM, less than about 20 pM, orless than about 10 pM, as measured by surface plasmon resonance, e.g.,using an assay format as defined in Example 3 herein (e.g., mAb-captureor antigen-capture format), or a substantially similar assay.

In some embodiments, the CD3-binding domain exhibits an EC₅₀ value ofless than less than about 50 nM, less than about 40 nM, less than about30 nM, less than about 20 nM, less than about 10 nM, less than about 5nM, less than about 4 nM, less than about 3 nM, less than about 2 nM,less than about 1 nM, less than 900 pM, less than 800 pM, less than 700pM, less than 600 pM, or less than 500 pM, as measured in an in vitroflow cytometry binding assay. In some embodiments, the CD3-bindingdomain exhibits an EC₅₀ value of about or greater than about 1 nM, 2 nM,3 nM, 4 nM, 5 nM, 10 nM, 25 nM, 50 nM, 100 nM, 500 nM or 1 μM, asmeasured in an in vitro flow cytometry binding assay.

In any of the embodiments, the CD3-binding domain can comprise any ofthe HCVR/LCVR or CDR (e.g., the six CDRs contained within a pair ofHCVR/LCVR sequences) amino acid sequences of the anti-CD3 antibodiesdisclosed in WO 2014/047231 (9250-WO) or WO 2017/053856 (10151WO01),including the antibodies identified as 7195P, 7221G, 7221G5 and 7221G20.In various embodiments, an anti-CD3 antibody identified as a “strongbinder” has an affinity for human CD3 in the single digit nanomolarrange (e.g., from 1-9 nM) as measured in a surface plasmon resonanceassay (e.g., at 25° C. in an antigen-capture format with measurementsconducted on a T200 BIACORE instrument). In various embodiments, ananti-CD3 antibody identified as a “moderate binder” has an affinity forhuman CD3 in the double digit nanomolar range (e.g., from 10-99 nM,optionally from 10-50 nM or 10-25 nM) as measured in a surface plasmonresonance assay. In various embodiments, an anti-CD3 antibody identifiedas a “weak binder” has an affinity for human CD3 in the three digitnanomolar range (e.g., from 100-999 nM, optionally from 100-500 nM orfrom 500 nM to 1 μM) as measured in a surface plasmon resonance assay.In various embodiments, an anti-CD3 antibody identified as a “very weakbinder” has an affinity for human CD3 that is greater than 10 μM or isundetectable as measured in a surface plasmon resonance assay.

In any of the embodiments, the CD3-binding domain can comprise any ofthe HCVR/LCVR or CDR (e.g., the six CDRs contained within a pair ofHCVR/LCVR sequences) amino acid sequences set forth in the followingtables (the “G” versions are taken from WO 2017/053856) In someembodiments, the CD3-binding domains (e.g., in the Fab arm of a moleculehaving the structure of FIG. 1C or 1F) comprise a cognate light chaincorresponding to the target antigen binding domain. In other words, thecognate light chain of the target antigen binding domain is common toboth the target antigen-binding domain and the CD3-binding domain (e.g.,in the N-terminal Fab domains of the structure of FIG. 1C or 1F).

TABLE 1 Heavy Chain Amino Acid Sequence Identifiers Antibody CD3-VH SEQID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 2 4 6 8 CD3-VH-G2 10 1214 16 CD3-VH-G3 18 20 22 24 CD3-VH-G4 26 28 30 32 CD3-VH-G5 34 36 38 40CD3-VH-G8 42 44 46 48 CD3-VH-G9 50 52 54 56 CD3-VH-G10 58 60 62 64CD3-VH-G11 66 68 70 72 CD3-VH-G12 74 76 78 80 CD3-VH-G13 82 84 86 88CD3-VH-G14 90 92 94 96 CD3-VH-G15 98 100 102 104 CD3-VH-G16 106 108 110112 CD3-VH-G17 114 116 118 120 CD3-VH-G18 122 124 126 128 CD3-VH-G19 130132 134 136 CD3-VH-G20 138 140 142 144 CD3-VH-G21 146 148 150 152 7195P154 156 158 160

TABLE 2 Heavy Chain Nucleic Acid Sequence Identifiers Antibody CD3-VHSEQ ID NOs: Designation HCVR CDR1 CDR2 CDR3 CD3-VH-G 1 3 5 7 CD3-VH-G2 911 13 15 CD3-VH-G3 17 19 21 23 CD3-VH-G4 25 27 29 31 CD3-VH-G5 33 35 3739 CD3-VH-G8 41 43 45 47 CD3-VH-G9 49 51 53 55 CD3-VH-G10 57 59 61 63CD3-VH-G11 65 67 69 71 CD3-VH-G12 73 75 77 79 CD3-VH-G13 81 83 85 87CD3-VH-G14 89 91 93 95 CD3-VH-G15 97 99 101 103 CD3-VH-G16 105 107 109111 CD3-VH-G17 113 115 117 119 CD3-VH-G18 121 123 125 127 CD3-VH-G19 129131 133 135 CD3-VH-G20 137 139 141 143 CD3-VH-G21 145 147 149 151 7195P153 155 157 159

TABLE 3 Light Chain Amino Acid Sequence Identifiers Antibody ULC SEQ IDNOs: Designation LCVR CDR1 CDR2 CDR3 Vκ1-39JK5 162 164 166 168

TABLE 4 Light Chain Nucleic Acid Sequence Identifiers Antibody ULC SEQID NOs: Designation LCVR CDR1 CDR2 CDR3 Vκ1-39JK5 161 163 165 167

Each of the antibodies set forth in Table 1 comprises a common lightchain variable region comprising the amino acid sequence set forth inTable 3. Each of the “G” designated antibodies may also be referred toherein with a “7221” prefix, e.g., 7221G, 7221G5, 7221G20, etc. In thescFv versions of the antigen-binding domains, the amino acid residue atposition 44 of the heavy chain variable region may be replaced with acysteine residue, for example, as shown in SEQ ID NO: 169 (the modifiedheavy chain corresponding to 7195P) or SEQ ID NO: 170 (the modifiedheavy chain corresponding to 7221G).

The multispecific antigen-binding molecules (e.g., bispecific ortrispecific or tetraspecific) of the present invention comprise twopolypeptide chains, each of which includes a multimerizing domain thatfacilitates association of the two polypeptide chains (e.g., viainterchain disulfide bonding) to form a single multispecificantigen-binding molecule. In any of the embodiments discussed above orherein, the first and second multimerizing domains can be immunoglobulinFc domains (e.g. of human IgG isotype). In some cases, the first andsecond multimerizing domains associate with one another via disulfidebonding. In some embodiments, the first multimerizing domain and thesecond multimerizing domain are human IgG1 or human IgG4 Fc domains. Insome cases, the first and second multimerizing domains comprise a hingedomain, a CH2 domain and a CH3 domain of human IgG1 or human IgG4.

In some embodiments, the first multimerizing domain or the secondmultimerizing domain comprises an amino acid substitution that reducesaffinity for Protein A binding compared to a wild-type Fc domain of thesame isotype (e.g., human IgG1 or human IgG4). In some cases, the aminoacid substitution comprises an H435R modification, or H435R and Y436Fmodifications (EU numbering). In some cases, the first multimerizingdomain comprises the H435R and Y436F modifications. In some cases, thesecond multimerizing domain comprises the H435R and Y436F modifications.

In some embodiments, the first polypeptide, the second polypeptide, orboth the first and the second polypeptides comprise a modified hingedomain that reduces binding affinity for an Fcγ receptor relative to awild-type hinge domain of the same isotype (e.g., human IgG1 or humanIgG4).

In various embodiments in which the multimerizing domain comprises aheavy chain constant region including a hinge domain, the constantregion may be chimeric, combining sequences derived from more than oneimmunoglobulin isotype. For example, a chimeric Fc domain can comprisepart or all of a C_(H)2 sequence derived from a human IgG1, human IgG2or human IgG4 C_(H)2 region, and part or all of a C_(H)3 sequencederived from a human IgG1, human IgG2 or human IgG4. A chimeric Fcdomain can also contain a chimeric hinge region. For example, a chimerichinge may comprise an “upper hinge” sequence, derived from a human IgG1,a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge”sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hingeregion. A particular example of a chimeric Fc domain that can beincluded in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fcdomain that can be included in any of the antigen-binding molecules setforth herein comprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upperhinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and otherexamples of chimeric Fc domains that can be included in any of theantigen-binding molecules of the present invention are described in WO2014/121087 (8550-WO). Chimeric Fc domains having these generalstructural arrangements, and variants thereof, can have altered Fcreceptor binding, which in turn affects Fc effector function.

In various embodiments in which the multimerizing domain comprises aheavy chain constant region including a hinge domain, positions 233-236within the hinge domain may be G, G, G and unoccupied; G, G, unoccupied,and unoccupied; G, unoccupied, unoccupied, and unoccupied; or allunoccupied, with positions numbered by EU numbering. Optionally, theheavy chain constant region comprises from N-terminal to C-terminal thehinge domain, a CH2 domain and a CH3 domain. Optionally, the heavy chainconstant region comprises from N-terminal to C-terminal a CH1 domain,the hinge domain, a CH2 domain and a CH3 domain. Optionally, the CH1region, if present, remainder of the hinge region, if any, CH2 regionand CH3 region are the same human isotype. Optionally, the CH1 region,if present, remainder of the hinge region, if any, CH2 region and CH3region are human IgG1. Optionally, the CH1 region, if present, remainderof the hinge region, if any, CH2 region and CH3 region are human IgG2.Optionally, the CH1 region if present, remainder of the hinge region, ifany, CH2 region and CH3 region are human IgG4. Optionally, the constantregion has a CH3 domain modified to reduce binding to protein A. Theseand other examples of multimerizing heavy chain constant regions thatcan be included in any of the antigen-binding molecules of the presentinvention are described in WO 2016/161010 (10140WO01).

In embodiments of the present invention, the association of onemultimerizing domain with another multimerizing domain facilitates theassociation between the two antigen-binding domains, thereby forming amultispecific antigen-binding molecule. The multimerizing domain may beany macromolecule, protein, polypeptide, peptide, or amino acid that hasthe ability to associate with a second multimerizing domain of the sameor similar structure or constitution. For example, a multimerizingdomain may be a polypeptide comprising an immunoglobulin C_(H)3 domain.A non-limiting example of a multimerizing component is an Fc portion ofan immunoglobulin (comprising a C_(H)2-C_(H)3 domain), e.g., an Fcdomain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4,as well as any allotype within each isotype group.

In some embodiments, the first and second multimerizing domains may beof the same IgG isotype such as, e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4.Alternatively, the first and second multimerizing domains may be ofdifferent IgG isotypes such as, e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4,etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of from 1 to about 200 amino acids in lengthcontaining at least one cysteine residue. In other embodiments, themultimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

The multimerizing domains, e.g., Fc domains (with or without a hinge),may comprise one or more amino acid changes (e.g., insertions, deletionsor substitutions) as compared to the wild-type, naturally occurringversion of the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., UY/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D orT); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q or K)and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 2591 (e.g., V2591), and308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g.,434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes multispecific antigen-bindingmolecules comprising a first Ig C_(H)3 domain and a second Ig C_(H)3domain, wherein the first and second Ig C_(H)3 domains differ from oneanother by at least one amino acid, and wherein at least one amino aciddifference reduces binding of the bispecific antibody to Protein A ascompared to a bi-specific antibody lacking the amino acid difference. Inone embodiment, the first Ig C_(H)3 domain binds Protein A and thesecond Ig C_(H)3 domain contains a mutation that reduces or abolishesProtein A binding such as an H95R modification (by IMGT exon numbering;H435R by EU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No.8,586,713. Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,different antigen-binding domains, specific for two or more differentantigens (e.g., CD3 and a target antigen), can be appropriately arrangedrelative to one another to produce the structures of the multispecificantigen-binding molecules of the present invention using routinemethods. In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains or parts thereof) of themultispecific antigen-binding molecules of the invention are derivedfrom chimeric, humanized or fully human antibodies. Methods for makingsuch antibodies are well known in the art. For example, one or more ofthe heavy and/or light chains of the multispecific antigen-bindingmolecules of the present invention can be prepared using VELOCIMMUNE™technology. Using VELOCIMMUNE™ technology (or any other human antibodygenerating technology), high affinity chimeric antibodies to aparticular antigen (e.g., CD3 or a target antigen) are initiallyisolated having a human variable region and a mouse constant region. Theantibodies are characterized and selected for desirable characteristics,including affinity, selectivity, epitope, etc. The mouse constantregions are replaced with a desired human constant region to generatefully human heavy and/or light chains that can be incorporated into themultispecific antigen-binding molecules of the present invention.

Genetically engineered animals may be used to make human multispecificantigen-binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human multispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454). Fully human refers to anantibody, or antigen-binding fragment or immunoglobulin domain thereof,comprising an amino acid sequence encoded by a DNA derived from a humansequence over the entire length of each polypeptide of the antibody orantigen-binding fragment or immunoglobulin domain thereof. In someinstances, the fully human sequence is derived from a protein endogenousto a human. In other instances, the fully human protein or proteinsequence comprises a chimeric sequence wherein each component sequenceis derived from human sequence. While not being bound by any one theory,chimeric proteins or chimeric sequences are generally designed tominimize the creation of immunogenic epitopes in the junctions ofcomponent sequences, e.g. compared to any wild-type human immunoglobulinregions or domains.

In various embodiments, the methods and techniques discussed above areused to generate antibodies to a T-cell antigen and a target antigen,and the antigen-binding domains of these antibodies (e.g., the HCVR,LCVR, or CDRs) are used to produce the multispecific antigen-bindingmolecules as discussed herein or having, e.g., the structuresillustrated in FIGS. 1C and 1E-1S.

Binding Properties of the Antigen-Binding Domains

As used herein, the term “binding” in the context of the binding of anantibody (e.g., a corresponding antibody), immunoglobulin,antigen-binding domain or multispecific antigen-binding molecule to,e.g., a predetermined antigen, such as a cell surface protein orfragment thereof, typically refers to an interaction or associationbetween a minimum of two entities or molecular structures, such as anantigen-binding domain/antigen interaction.

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore 3000 instrument using the antigen as theligand and the antibody, Ig, antibody-binding domain or multispecificantigen-binding molecule as the analyte (or anti-ligand). Flow cytometryassays are also routinely used.

Accordingly, the antibody (e.g., a corresponding antibody),antigen-binding domain or multispecific antigen-binding molecule of theinvention binds to the predetermined antigen or cell surface moleculehaving an affinity corresponding to a K_(D) value that is at leastten-fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein). According to the present invention, the affinity ofan antibody (e.g., a corresponding antibody), antigen-binding domain ormultispecific antigen-binding molecule corresponding to a K_(D) valuethat is equal to or less than ten-fold lower than a non-specific antigenmay be considered non-detectable binding, however such an antibody maybe paired with a second antigen binding arm for the production of abispecific antibody of the invention.

The term “K_(D)” (M) refers to the dissociation equilibrium constant ofa particular antibody (or antigen-binding domain)-antigen interaction,or the dissociation equilibrium constant of an antibody (orantigen-binding domain) or antibody-binding fragment binding to anantigen. There is an inverse relationship between K_(D) and bindingaffinity, therefore the smaller the K_(D) value, the higher, i.e.stronger, the affinity. Thus, the terms “higher affinity” or “strongeraffinity” relate to a higher ability to form an interaction andtherefore a smaller K_(D) value, and conversely the terms “loweraffinity” or “weaker affinity” relate to a lower ability to form aninteraction and therefore a larger K_(D) value. In some circumstances, ahigher binding affinity (or K_(D)) of a particular molecule (e.g.antibody or antigen-binding domain) to its interactive partner molecule(e.g. antigen X) compared to the binding affinity of the molecule (e.g.antibody or antigen-binding domain) to another interactive partnermolecule (e.g. antigen Y) may be expressed as a binding ratio determinedby dividing the larger K_(D) value (lower, or weaker, affinity) by thesmaller K_(D) (higher, or stronger, affinity), for example expressed as5-fold or 10-fold greater binding affinity, as the case may be.

The term “k_(d)” (sec−1 or 1/s) refers to the dissociation rate constantof a particular antibody (or antigen-binding domain)-antigeninteraction, or the dissociation rate constant of an antibody orantibody-binding domain. Said value is also referred to as the k_(off)value.

The term “k_(a)” (M−1×sec−1 or 1/M) refers to the association rateconstant of a particular antibody (or antigen-binding domain)-antigeninteraction, or the association rate constant of an antibody orantibody-binding domain.

The term “K_(A)” (M−1 or 1/M) refers to the association equilibriumconstant of a particular antibody (or antigen-binding domain)-antigeninteraction, or the association equilibrium constant of an antibody orantibody-binding domain. The association equilibrium constant isobtained by dividing the k_(a) by the k_(d).

The term “EC50” or “EC₅₀” refers to the half maximal effectiveconcentration, which includes the concentration of an antibody (orantigen-binding domain or multispecific molecule) which induces aresponse halfway between the baseline and maximum after a specifiedexposure time. The EC₅₀ essentially represents the concentration of anantibody (or antigen-binding domain or multispecific molecule) where 50%of its maximal effect is observed. In certain embodiments, the EC₅₀value equals the concentration of a multispecific molecule of theinvention that gives half-maximal binding to cells expressing CD3 ortarget antigen (e.g., tumor-associated antigen), as determined by e.g. aflow cytometry binding assay. Thus, reduced or weaker binding isobserved with an increased EC₅₀, or half maximal effective concentrationvalue.

In one embodiment, decreased binding can be defined as an increased EC₅₀molecule concentration which enables binding to the half-maximal amountof target cells.

In another embodiment, the EC₅₀ value represents the concentration of amolecule of the invention that elicits half-maximal depletion of targetcells by T cell cytotoxic activity. Thus, increased cytotoxic activity(e.g. T cell-mediated tumor cell killing) is observed with a decreasedEC₅₀, or half maximal effective concentration value.

pH-Dependent Binding

The present invention includes antigen-binding domains and multispecificantigen-binding molecules with pH-dependent binding characteristics. Forexample, a molecule of the present invention may exhibit reduced bindingto a T-cell antigen or a target antigen at acidic pH as compared toneutral pH. Alternatively, molecules of the invention may exhibitenhanced binding to a T-cell antigen or a target antigen at acidic pH ascompared to neutral pH. The expression “acidic pH” includes pH valuesless than about 6.2, e.g., about 6.0, 5.95, 5,9, 5.85, 5.8, 5.75, 5.7,5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05,5.0, or less. As used herein, the expression “neutral pH” means a pH ofabout 7.0 to about 7.4. The expression “neutral pH” includes pH valuesof about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding . . . at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of themolecule (or antigen-binding domain) binding to its antigen at acidic pHto the K_(D) value of the molecule (or antigen-binding domain) bindingto its antigen at neutral pH (or vice versa). For example, a molecule orantigen-binding domain may be regarded as exhibiting “reduced binding toa T-cell antigen or a target antigen at acidic pH as compared to neutralpH” for purposes of the present invention if the molecule orantigen-binding domain exhibits an acidic/neutral K_(D) ratio of about3.0 or greater. In certain exemplary embodiments, the acidic/neutralK_(D) ratio for a molecule or antigen-binding domain of the presentinvention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5,14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0 orgreater.

Multispecific molecules with pH-dependent binding characteristics may beobtained, e.g., by screening a population of corresponding antibodiesfor reduced (or enhanced) binding to a particular antigen at acidic pHas compared to neutral pH. Additionally, modifications of theantigen-binding domain at the amino acid level may yield molecules withpH-dependent characteristics. For example, by substituting one or moreamino acids of an antigen-binding domain (e.g., within a CDR) with ahistidine residue, a molecule with reduced antigen-binding at acidic pHrelative to neutral pH may be obtained.

Biological Characteristics of the Multispecific Antigen-BindingMolecules

The present invention can include multispecific antigen-bindingmolecules and antigen-binding domains thereof that are capable ofsimultaneously binding to a human T-cell antigen (e.g., CD3) and a humantarget antigen or antigens (e.g., a tumor-associated antigen).

The present invention can include multispecific antigen-bindingmolecules that bind a human T-cell antigen (e.g., CD3) and induce T cellactivation in the presence of target cells. For example, in someembodiments, the present invention includes multispecificantigen-binding molecules that bind a human T-cell antigen (e.g., CD3)and induce T cell cytotoxic activity in the presence of cells expressingthe target antigen or target antigens (e.g., a tumor-associatedantigen).

The present invention can include multispecific antigen-bindingmolecules that bind a human T-cell antigen (e.g., CD3) and induce T cellactivation without increasing cytokine production relative to aconventionally structured bispecific anti-CD3 x anti-TA antibody (e.g.,FIG. 1A).

The present invention can include multispecific antigen-bindingmolecules that are capable of depleting or reducing cell populations inwhich the cells express the target antigen or target antigens. Themultispecific antigen-binding molecules of the present invention arecapable of inducing T-cell mediated cytotoxicity more potently thanmolecules having conventional bispecific antibody formats (e.g., FIGS.1A and 1B).

The present invention can include multispecific antigen-bindingmolecules that bind a human T-cell antigen (e.g., CD3) and two distincttarget antigens (e.g., a molecule having the structure of FIG. 1F), andinduce cytotoxic activity and/or T-cell activation in the presence ofcells expressing the two target antigens.

Many cancers express a variety of intracellular antigens that areprocessed inside the cell by the proteosome and associated peptides arepresented at the surface of the cell in the context of HLA molecules.Targeting peptides from different proteins may be used to increase thespecificity of the multispecific molecules of the present invention. Insome cases, cancers characterized by PiG antigens or low density cancerantigens escape conventional cancer therapies because they are oftenpresent in low target copy numbers within tumors. Additionally, solidtumors characterized by PiGs or low density cancer antigens can be moreresistant to therapy and more difficult to treat because they are notcell surface antigens, but are present in grooves within the cancerrelated peptide. Thus, use of a multispecific molecule of the presentinvention targeting two distinct antigens (e.g., low density antigens)can effectively target PiGs and/or low density cancer antigens toincrease/enhance efficacy of therapy in cancers, especially thosecancers characterized by solid tumors.

In various embodiments, the multispecific antigen-binding molecules ofthe present invention are capable of inducing T-cell mediatedcytotoxicity in cell populations when the density of the target antigenranges from about 100 copies per cell to about 1 million copies per cellor more. In some cases, the target antigen is present at a copynumber/cell of about 100, about 200, about 300, about 400, about 500,about 1000, about 2000, about 3000, about 4000, about 5000, about 6000,about 7000, about 8000, about 9000, about 10000, about 15000, about20000, about 25000, about 30000, about 35000, about 40000, about 45000,about 50000, about 75000, about 100000 (i.e., 100K), about 200K, about300K, about 400K, about 500K, about 600K, about 700K, about 800K, about900K, about 1 million, about 2 million, about 3 million, about 4million, about 5 million, or about 10 million.

Without intending to be bound by theory, the inventors postulate thatthe improved cytotoxic potency of the molecular format of the presentinvention is a function of the presence of two T-cell antigen (e.g.,CD3) binding domains on a single chain of the molecule. In particular,it is hypothesized that the geometry of the molecular structures of thepresent invention selectively induces lytic synapse formation at lowconcentrations without inducing stimulatory synapse formation, thelatter of which is responsible for cytokine production from cytotoxic Tlymphocytes.

Epitope Mapping and Related Technologies

The epitope on the T-cell antigen (e.g., CD3) and/or the target antigen(e.g., a tumor-associated antigen) to which the antigen-bindingmolecules of the present invention bind may consist of a singlecontiguous sequence of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of a protein.Alternatively, the epitope may consist of a plurality of non-contiguousamino acids (or amino acid sequences) of the protein. The molecules ofthe invention may interact with, e.g., amino acids contained within asingle CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma), or mayinteract with amino acids on two or more different CD3 chains. The term“epitope,” as used herein, refers to an antigenic determinant thatinteracts with a specific antigen binding site in the variable region ofan antigen-binding domain known as a paratope. A single antigen may havemore than one epitope. Thus, different antigen-binding domains may bindto different areas on an antigen and may have different biologicaleffects. Epitopes may be either conformational or linear. Aconformational epitope is produced by spatially juxtaposed amino acidsfrom different segments of the linear polypeptide chain. A linearepitope is one produced by adjacent amino acid residues in a polypeptidechain. In certain circumstances, an epitope may include moieties ofsaccharides, phosphoryl groups, or sulfonyl groups on the antigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of a molecule“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques include, e.g., routine cross-blockingassay such as that described Antibodies, Harlow and Lane (Cold SpringHarbor Press, Cold Spring Harb., N.Y.), alanine scanning mutationalanalysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol248:443-463), and peptide cleavage analysis. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer, 2000, Protein Science 9:487-496).Another method that can be used to identify the amino acids within apolypeptide with which an antigen-binding domain of a molecule interactsis hydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding themolecule to the deuterium-labeled protein. Next, the protein/moleculecomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the molecule(which remain deuterium-labeled). After dissociation of the molecule,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the moleculeinteracts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-raycrystallography of the antigen/molecule complex may also be used forepitope mapping purposes.

Bioequivalents

The present invention includes multispecific antigen-binding moleculesthat are bioequivalent to any of the exemplary multispecificantigen-binding molecules set forth herein. Two antigen-binding proteinsare considered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledoes or multiple dose. Some antigen-binding proteins will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antigen-binding proteinor its metabolites is measured in blood, plasma, serum, or otherbiological fluid as a function of time; (b) an in vitro test that hasbeen correlated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of theantigen-binding protein (or its target) is measured as a function oftime; and (d) in a well-controlled clinical trial that establishessafety, efficacy, or bioavailability or bioequivalence of anantigen-binding protein.

Bioequivalent variants of the exemplary multispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antigen-binding proteinsmay include variants of the exemplary multispecific antigen-bindingmolecules set forth herein comprising amino acid changes which modifythe glycosylation characteristics of the molecules, e.g., mutationswhich eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, antigen-bindingmolecules are provided which bind to human T cell antigen (e.g., CD3)but not to the same antigen from other species. Also provided areantigen-binding molecules which bind to human target antigens (e.g.,tumor antigens) but not to the same target antigens from other species.The present invention also includes antigen-binding molecules that bindto human antigens and corresponding antigens from one or more non-humanspecies.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provided which bind to human CD3 and/or ahuman tumor antigen and may bind or not bind, as the case may be, to oneor more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog,rabbit, goat, sheep, cow, horse, camel, cynomolgus, marmoset, rhesus orchimpanzee CD3 and/or tumor antigen. For example, in a particularexemplary embodiment of the present invention, multispecificantigen-binding molecules are provided comprising a firstantigen-binding domain that binds human CD3 and cynomolgus CD3, and asecond antigen-binding domain that specifically binds a human tumorantigen.

Immunoconjugates

The present invention encompasses antigen-binding molecules conjugatedto a therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, (see for example, WO 05/103081).

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe multispecific antigen-binding molecules of the present invention.The pharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area. Whena multispecific antigen-binding molecule of the present invention isused for therapeutic purposes in an adult patient, it may beadvantageous to intravenously administer the multispecificantigen-binding molecule of the present invention normally at a singledose of about 0.01 to about 20 mg/kg body weight, more preferably about0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg/kgbody weight. Depending on the severity of the condition, the frequencyand the duration of the treatment can be adjusted. Effective dosages andschedules for administering a multispecific antigen-binding molecule maybe determined empirically; for example, patient progress can bemonitored by periodic assessment, and the dose adjusted accordingly.Moreover, interspecies scaling of dosages can be performed usingwell-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut.Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pens and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Florida. In yet another embodiment, a controlledrelease system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying theantigen-binding molecule or its salt described above in a sterileaqueous medium or an oily medium conventionally used for injections. Asthe aqueous medium for injections, there are, for example, physiologicalsaline, an isotonic solution containing glucose and other auxiliaryagents, etc., which may be used in combination with an appropriatesolubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol(e.g., propylene glycol, polyethylene glycol), a nonionic surfactant[e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct ofhydrogenated castor oil)], etc. As the oily medium, there are employed,e.g., sesame oil, soybean oil, etc., which may be used in combinationwith a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc.The injection thus prepared is preferably filled in an appropriateampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaidantigen-binding molecule contained is generally about 5 to about 500 mgper dosage form in a unit dose; especially in the form of injection, itis preferred that the aforesaid antigen-binding molecule is contained inabout 5 to about 100 mg and in about 10 to about 250 mg for the otherdosage forms.

Therapeutic uses of the Antigen-Binding Molecules

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising amultispecific antigen-binding molecule that specifically binds a T-cellantigen (e.g., CD3) and a target antigen (e.g., a tumor-associatedantigen). The therapeutic composition can comprise any of themultispecific antigen-binding molecules as disclosed herein and apharmaceutically acceptable carrier or diluent. As used herein, theexpression “a subject in need thereof” means a human or non-human animalthat exhibits one or more symptoms or indicia of cancer, or whootherwise would benefit from an inhibition or reduction in targetantigen activity or a depletion of target-antigen positive cells (e.g.,tumor cells).

The multispecific antigen-binding molecules of the invention (andtherapeutic compositions comprising the same) are useful, inter alia,for treating any disease or disorder in which stimulation, activationand/or targeting of an immune response would be beneficial. Inparticular, the multispecific antigen-binding molecules of the presentinvention may be used for the treatment, prevention and/or ameliorationof any disease or disorder associated with or mediated by target antigenexpression or activity or the proliferation of target-antigen positivecells. The mechanism of action by which the therapeutic methods of theinvention are achieved includes killing of the cells expressing thetarget antigen in the presence of T cells.

The multispecific antigen-binding molecules of the present invention maybe used to treat a disease or disorder associated with target antigenexpression including, e.g., a cancer. Analytic/diagnostic methods knownin the art, such as tumor scanning, etc., may be used to ascertainwhether a patient harbors a tumor cell that is positive for the targetantigen. In some cases, the cancer is selected from a solid tumor,cervical cancer, head and neck squamous cell carcinoma, melanoma,prostate cancer, acute myeloid leukemia, pancreatic cancer, coloncancer, acute lymphocytic leukemia, a non-Hodgkin's lymphoma, gastriccancer, post-transplant lymphoproliferative disorder, ovarian cancer,lung cancer, squamous cell carcinoma, non-small cell lung canceresophageal cancer, bladder cancer, nasopharyngeal cancer, uterinecancer, liver cancer, testicular cancer, or breast cancer.

The present invention also includes methods for treating residual cancerin a subject. As used herein, the term “residual cancer” means theexistence or persistence of one or more cancerous cells in a subjectfollowing treatment with an anti-cancer therapy.

According to certain aspects, the present invention provides methods fortreating a disease or disorder associated with target antigen expression(e.g., a cancer) comprising administering one or more of themultispecific antigen-binding molecules described elsewhere herein to asubject after the subject has been determined to have a target antigenpositive cancer. For example, the present invention includes methods fortreating a cancer comprising administering a multispecificantigen-binding molecule to a patient 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks, 2 months, 4 months, 6months, 8 months, 1 year, or more after the subject has received otherimmunotherapy or chemotherapy.

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary multispecificantigen-binding molecules described herein in combination with one ormore additional therapeutic agents. Exemplary additional therapeuticagents that may be combined with or administered in combination with anantigen-binding molecule of the present invention include, e.g., ananti-tumor agent (e.g. chemotherapeutic agents). In certain embodiments,the second therapeutic agent may be a monoclonal antibody, an antibodydrug conjugate, a bispecific antibody conjugated to an anti-tumor agent,a checkpoint inhibitor, or combinations thereof. Other agents that maybe beneficially administered in combination with the antigen-bindingmolecules of the invention include cytokine inhibitors, includingsmall-molecule cytokine inhibitors and antibodies that bind to cytokinessuch as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-17, IL-18, or to their respective receptors. Thepharmaceutical compositions of the present invention (e.g.,pharmaceutical compositions comprising a multispecific antigen-bindingmolecule as disclosed herein) may also be administered as part of atherapeutic regimen comprising one or more therapeutic combinationsselected from a monoclonal antibody that may interact with a differentantigen on the cell surface, a bispecific antibody that has one arm thatbinds to an antigen on the tumor cell surface and the other arm binds toan antigen on a T cell, an antibody drug conjugate, a bispecificantibody conjugated with an anti-tumor agent, a checkpoint inhibitor,for example, one that targets, PD-1 or CTLA-4, or combinations thereof.In certain embodiments, the checkpoint inhibitors may be selected fromPD-1 inhibitors, such as pembrolizumab (Keytruda), nivolumab (Opdivo),or cemiplimab (REGN2810). In certain embodiments, the checkpointinhibitors may be selected from PD-L1 inhibitors, such as atezolizumab(Tecentriq), avelumab (Bavencio), or Durvalumab (Imfinzi)). In certainembodiments, the checkpoint inhibitors may be selected from CTLA-4inhibitors, such as ipilimumab (Yervoy). Other combinations that may beused in conjunction with an antibody of the invention are describedabove.

The present invention also includes therapeutic combinations comprisingany of the antigen-binding molecules mentioned herein and an inhibitorof one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII,cMet, IGF1R, IL-10, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1,STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementionedcytokines, wherein the inhibitor is an aptamer, an antisense molecule, aribozyme, an siRNA, a peptibody, a nanobody, an antibody, a bispecificantibody or an antibody fragment (e.g., Fab fragment; F(ab′)₂ fragment;Fd fragment; Fv fragment; scFv; dAb fragment; or other engineeredmolecules, such as diabodies, triabodies, tetrabodies, minibodies andminimal recognition units). The antigen-binding molecules of theinvention may also be administered and/or co-formulated in combinationwith antivirals, antibiotics, analgesics, corticosteroids and/or NSAIDs.The antigen-binding molecules of the invention may also be administeredas part of a treatment regimen that also includes radiation treatmentand/or conventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan antigen-binding molecule of the present invention; (for purposes ofthe present disclosure, such administration regimens are considered theadministration of an antigen-binding molecule “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which anantigen-binding molecule of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of a multispecific antigen-binding molecule may be administered toa subject over a defined time course. The methods according to thisaspect of the invention comprise sequentially administering to a subjectmultiple doses of an antigen-binding molecule of the invention. As usedherein, “sequentially administering” means that each dose of anantigen-binding molecule is administered to the subject at a differentpoint in time, e.g., on different days separated by a predeterminedinterval (e.g., hours, days, weeks or months). The present inventionincludes methods which comprise sequentially administering to thepatient a single initial dose of an antigen-binding molecule, followedby one or more secondary doses of the antigen-binding molecule, andoptionally followed by one or more tertiary doses of the antigen-bindingmolecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of an antigen-binding molecule contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule. For example, in certain embodiments,only a single secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Method for Binding by Flow Cytometry: In the following examples, bindingfor the various molecules was determined using the following flowcytometry method. Flow cytometric analysis was utilized to determinebinding of MAGEA4 x CD3 multispecific molecules toRAJI/HLA-A2/B2M/MAGEA4(peptide a), A375/hHLA-A2/B2M/MAGEA4(peptide b),RAJI/HLA-A2/B2M/NY-ESO-1, and JURKAT cells, followed by detection withan APC-labeled anti-human IgG antibody. Briefly, 1×10⁵ cells/well wereincubated for 30 minutes at 4° C. with a serial dilution of MAGEA4 x CD3multispecific molecules or Isotype control (a human IgG4 stealthantibody that binds a human antigen with no cross-reactivity to humanMAGEA4 or CD3). After incubation, the cells were washed twice with coldPBS containing 1% filtered FBS and a PE-conjugated anti-human secondaryantibody was added to the cells and incubated for an additional 30minutes. Wells containing no antibody or secondary only were used as acontrol. After incubation, cells were washed, re-suspended in 200 μLcold PBS containing 1% filtered FBS and analyzed by flow cytometry on aBD FACS Canto II.

Method for Cytotoxicity Assay: In the following examples, cytotoxicityof the various molecules was determined using the following cytotoxicityassay. In order to monitor the killing of MAGEA4+ cells in the presenceof MAGEA4 x CD3 as single agents or in combination with an EGFR x CD28bispecific antibody and/or a PD-1 antibody, A375 cells, ScaBER cells,NCI-H1755 metastatic (from liver) cells, and NCI-H1755 cells werelabeled with 1 μM of the fluorescent tracking dye Violet Cell Tracker.After labeling, cells were plated overnight at 37° C. Separately, humanPBMCs were plated in supplemented RPMI media at 1×10⁶ cells/mL andincubated overnight at 37° C. in order to enrich for lymphocytes bydepleting adherent macrophages, dendritic cells, and some monocytes. Thenext day, target cells were co-incubated with adherent cell-depletednaïve PBMC (Effector/Target cell 10:1 ratio), a serial dilution ofMAGEA4 x CD3 multispecific molecules and a fixed concentration of EGFR xCD28 and/or anti-PD1 antibodies for 96 hours at 37° C. Cells wereremoved from cell culture plates using Trypsin-EDTA dissociation buffer,and analyzed by FACS on a FACS BD LSRFortessa-X20. For FACS analysis,cells were stained with a dead/live Near IR Reactive (Invitrogen) dye.5E05 counting beads were added to each well immediately before FACSanalysis. 1E05 beads were collected for each sample. For the assessmentof specificity of killing, cells were gated on live Violet labeledpopulations. Percent of live population was recorded and used for thecalculation of survival.

Example 1: T Cell Activation is Dependent on the Presence of TargetCells

T cell activation was evaluated for each of the molecular formatsillustrated in FIGS. 1A, 1B and 1C. T cell activation and upregulationof the PD-1 marker were assessed by incubating cells with directlyconjugated antibodies to CD2, CD4, CD8, CD25 and PD-1, and by reportingthe percent of late activated (CD25+/CD8+) T cells and PD-1+/CD4+ Tcells out of total T cells (CD2+).

As shown in FIG. 2, the exemplary multispecific molecule of the presentinvention (FIG. 1C structure) did not activate T cells in the absence oftarget cells. The “ZERO” represent a T cell only control.

Example 2: Cytotoxicity of Multispecific Molecules Relative toConventional Formats

Cytotoxicity of an exemplary multispecific molecule of the presentinvention (FIG. 1C structure) was measured as discussed above, andcompared to the cytotoxicity of conventionally formatted moleculeshaving the same antigen-binding domains (FIGS. 1A and 1B). TheCD3-binding domains used in this example have a moderate bindingaffinity to human CD3. The target antigen binding domain used in thisexample binds to a MAGEA4 (Melanoma-Associated Antigen A4) peptide. The“Control” is a positive control that targets the scaffold of all HLAmolecules to provide a maximum cytotoxicity against which to compare theother formats.

As illustrated in FIG. 3, the exemplary multispecific molecule of thepresent invention (FIG. 1C structure) more potently killed target cellsthan did the molecules having conventional bispecific formats (FIG. 1Astructure, and FIG. 1B structure).

Example 3: Cytotoxicity of Multispecific Molecules Relative toConventional Formats in Combination With an Anti-PD-1 Antibody, aCo-Stimulatory Bispecific Antibody, or Both

Cytotoxicity of an exemplary multispecific molecule of the presentinvention (FIG. 1C structure) was measured as discussed above, andcompared to the cytotoxicity of conventionally formatted moleculeshaving the same antigen-binding domains (FIGS. 1A and 1B) in combinationwith an anti-PD-1 antibody, a co-stimulatory bispecific EGFR x CD28antibody, or both an anti-PD-1 antibody and a costimulatory bispecificEGFR x CD28 antibody. The positive control, and the CD3 and targetantigen-binding domains were as discussed above in Example 2.

As illustrated in FIGS. 4A, 4B and 4C, the addition of an anti-PD-1antibody, a co-stimulatory bispecific EGFR x CD28 antibody, or both,further enhanced the potency of the exemplary multispecific molecule ofthe present invention (FIG. 1C structure). The solid lines represent thecytotoxicity of the single agent (as shown in FIG. 3), and the dashedlines represent the cytotoxicity of the respective combination.

In addition to cytotoxicity, the supernatant of the assay wells from thehuman PBMC assay were assessed for Th1/Th2 cytokine release using the BDcytometric bead array human kit and following the manufacturer'sprotocol. As illustrated in FIG. 5, the greater cytotoxicity of theexemplary multispecific molecules of the present invention (FIG. 1Cstructure) did not result in any greater cytokine release as compared tothe conventional bispecific antibody format (FIG. 1A structure).

This set of experiments confirms that: (a) at maximum concentration inthe cytotoxicity assay, the molecule having the structure of FIG. 1Cexhibited greater potency than did the molecule having the structure ofFIG. 1A with comparable levels of cytokine release; (b) the EC50 for thecytotoxicity of the molecule having the structure of FIG. 1C (singleagent) was lower than that observed for the molecule having thestructure of FIG. 1A (single agent); (c) at maximum concentration in thecytotoxicity assay, the molecule having the structure of FIG. 1Aexhibited greater potency in combination with an anti-PD-1 antibody thandid the molecule having the structure of FIG. 1A (anti-PD-1 combo) withcomparable levels of cytokine release; (d) the EC50 for the cytotoxicityof the molecule having the structure of FIG. 1C in combination with ananti-PD-1 antibody was lower than that observed for the molecule havingthe structure of FIG. 1A (anti-PD-1 combo); (e) at maximum concentrationin the cytotoxicity assay, the molecule having the structure of FIG. 1Aexhibited greater potency in combination with an anti-EGFR x CD28bispecific antibody than did the molecule having the structure of FIG.1A (anti-EGFR x CD28 combo) with comparable levels of cytokine release;(f) the EC50 for the cytotoxicity of the molecule having the structureof FIG. 1C in combination with an anti-EGFR x CD28 bispecific antibodywas lower than that observed for the molecule having the structure ofFIG. 1A (anti-EGFR x CD28 combo); (g) at maximum concentration in thecytotoxicity assay, the molecule having the structure of FIG. 1Aexhibited greater potency in combination with an anti-PD-1 antibody andan anti-EGFR x CD28 bispecific antibody than did the molecule having thestructure of FIG. 1A (triple combo) with comparable levels of cytokinerelease; (h) the EC50 for the cytotoxicity of the molecule having thestructure of FIG. 1C in combination with an anti-PD-1 antibody and ananti-EGFR x CD28 bispecific antibody was lower than that observed forthe molecule having the structure of FIG. 1A (triple combo).

Example 4: Potency of the Multispecific Molecules is Enhanced by TwoEffector Binding Domains

Binding of an exemplary multispecific molecule (FIG. 1C structure) totarget cells overexpressing a MAGEA4 peptide and CD3+ Jurkat cells wasmeasured as discussed above. Binding to these cells was also evaluatedfor modifications of the FIG. 1C structure in which one or more of theantigen-binding domains was made inactive. The inactive domains areillustrated with an “X” in the legend of the figures.

As illustrated in FIGS. 6A-6D, the binding data showed that thecombination of two antigen-binding domains (e.g., a single Fab and asingle scFv) bound to the target cells with greater affinity (lowerEC50) than did a molecule with a single Fab domain or molecule with asingle scFv domain. As expected, the isotype control molecule showed nobinding. No distinction in the binding pattern was observed irrespectiveof the source of the anti-CD3 binding domain.

In addition to binding, cytotoxicity of these molecules was alsodetermined using the method discussed above. As illustrated in FIGS. 7Aand 7B, the exemplary multispecific molecule (FIG. 1C structure) of thepresent invention showed the greatest cytotoxic potency, followed by thetwo modified molecules comprising two T-cell antigen (CD3) bindingdomains but only a single target antigen (MAGEA4) binding domain (scFvor Fab). Again, the same cytotoxic pattern was observed irrespective ofthe source of the anti-CD3 binding domain. The negative control (FIG. 1Aformat) comprised an irrelevant target antigen binding domain.

Example 5: C-Terminal scFv Domains Enhance Potency of the MultispecificMolecules Relative to C-Terminal Fab Domains

Binding of an exemplary multispecific molecule (FIG. 1C structure) totarget cells overexpressing a MAGEA4 peptide and CD3+ Jurkat cells wasmeasured as discussed above. Binding to these cells was also evaluatedfor modifications of the FIG. 1C structure to replace the C-terminalscFv domains with Fab domains (FIG. 1E structure), or in which theN-terminal Fab domains were made inactive. The inactive domains areillustrated with an “X” in the legend of the figures.

Similarly to the binding discussed in Example 4, and as illustrated inFIGS. 8A and 8B, the binding data showed that the combination of twoantigen-binding domains (e.g., a single Fab and a single scFv, or twoFabs) bound to the target cells with greater affinity (lower EC50) thandid a molecule with a single Fab domain or molecule with a single scFvdomain. As shown in the tables of FIGS. 8A and 8B, the molecules havingthe structures of FIG. 1C and FIG. 1E bind with comparable bindingtitrations.

In addition to binding, cytotoxicity of these molecules was alsodetermined using the method discussed above. As illustrated in FIG. 9,the exemplary multispecific molecule (FIG. 1C structure) of the presentinvention showed the greatest cytotoxic potency, followed by themodified molecule comprising C-terminal Fab domains in place of the twoscFv domains.

Example 6: Single Chain Bivalency for T-Cell Antigen Enhances Potency ofthe Multispecific Molecules Relative to Multiple Chain Bivalency

Binding of an exemplary multispecific molecule (FIG. 1C structure) totarget cells overexpressing a MAGEA4 peptide and CD3+ Jurkat cells wasmeasured as discussed above. Binding to these cells was also evaluatedfor the molecule having the structure illustrated in FIG. 1D, in whichthe MAGEA4-binding domain and the CD3-binding domain are swapped suchthat the two sets of antigen-binding domains are located on two separatepolypeptide chains.

As illustrated in FIGS. 10A and 10B, the binding data showed similarbinding of the two molecular structures to each of the two cell types.

In addition to binding, cytotoxicity of these molecules was alsodetermined using the method discussed above. As illustrated in FIGS. 11Aand 11B, the exemplary multispecific molecule (FIG. 1C structure) of thepresent invention showed the greater cytotoxic potency relative to themolecule having the structure of FIG. 1D, confirming that the presenceof two T-cell antigen binding domains on a single polypeptide chainprovides enhanced cytotoxic potency.

Example 7: Relative Cytotoxicity of Multispecific Molecules TargetingOne or Two Antigens Relative to Conventional Formats Alone or inCombination With an Anti-PD-1 Antibody and a Co-Stimulatory BispecificAntibody

Cytotoxicity of two exemplary multispecific molecules of the presentinvention (FIG. 1C and FIG. 1F structures) was measured as discussedabove, and compared to the cytotoxicity of a conventionally formattedmolecule (FIG. 1A), alone or in combination with an anti-PD-1 antibodyand a co-stimulatory bispecific EGFR x CD28 antibody. This example usesa positive control with greater specificity than that used in priorExamples to show the greater distinction between the molecules havingthe structures of FIGS. 1C and 1F, and the combinations of thesemolecules with the co-stimulatory bispecific antibody and the anti-PD-1antibody. The CD3 antigen-binding domains used in this example have astrong binding affinity to human CD3, and the target antigen-bindingdomains (MAGEA4a) were as discussed above in Example 2. The negativecontrol (FIG. 1A format) comprised an irrelevant target antigen bindingdomain. The second target antigen binding domain (MAGEA4b) used in thisexample for the molecule having the structure of FIG. 1F binds to anepitope of MAGEA4 that is completely distinct from the epitope bound bythe first target antigen binding domain.

As illustrated in FIGS. 12A and 12B, the multispecific molecule thattargets two different low density antigens on tumor cells showsincreased potency relative to the multispecific molecule that targetsonly a single tumor antigen, and both molecules show greater potencythan the conventionally formatted molecule having the structure of FIG.1A. The addition of an anti-PD-1 antibody and a co-stimulatorybispecific EGFR x CD28 antibody further enhanced the potency of theexemplary multispecific molecules of the present invention (FIGS. 1C and1F structures).

Example 8: Relative Cytotoxicity of Multispecific Molecules Correlatesto the Affinity of the T-Cell Antigen Binding Domain

Exemplary multispecific molecules having the structure of FIG. 1F (asshown in FIG. 13) were prepared with anti-CD3 binding domains of varyingaffinity. Five molecules were prepared according to the followingparameters:

Molecule A with CD3 arms 7195P (strong) fab and 7195P (strong) scfv;Molecule B with CD3 arms 7221G (moderate) fab and 7221G (moderate) scfv;Molecule C with CD3 arms 7221G20 (weak) fab and 7221G20 (weak) scfv;Molecule D with CD3 arms 7221G20 (weak) fab and 7221G (moderate) scfv;andMolecule E with CD3 arms 7221G (moderate) fab and 7195P (strong) scfv.

The range of binding titration to T cells from these five molecules wastested by flow cytometry, and correlates with the strength of the CD3binding domains, as shown in FIG. 13 relative to an isotype control.

In a cytotoxicity assay targeting two different MAGEA4+ cell lines (A375and ScaBER), the potency of the molecules was shown to decrease when thestrength of the effector arm (e.g., anti-CD3 binding domain) decreases,as single agent, or in combination with an EGFR x CD28 bispecificantibody and an anti-PD1 antibody, as shown in FIGS. 14A, 14B, 15A and15B. Each of the molecules contained the same target antigen bindingdomains (to non-overlapping MAGEA4 peptide 1 and MAGEA4 peptide 2).

Example 9: Relative Cytotoxicity of Multispecific Molecules TargetingTwo Antigens Relative to Conventional Formats Alone or in CombinationWith an Anti-PD-1 Antibody and a Co-Stimulatory Bispecific Antibody

Cytotoxicity of three exemplary multispecific molecules of the presentinvention (FIG. 1C and FIG. 1F structures) was measured as discussedabove, and compared to the cytotoxicity of a conventionally formattedmolecule (FIG. 1A), alone or in combination with an anti-PD-1 antibodyand a co-stimulatory bispecific EGFR x CD28 antibody. This example usesa positive control with the structure of FIG. 1A that binds CD3 and HLA.The CD3 antigen-binding domains used in this example have a strongbinding affinity to human CD3 (derived from 7195P), and the targetantigen-binding domains are to one or two non-overlapping MAGEA4(Melanoma-Associated Antigen A4) peptides (MAGEA4Aa and MAGEA4b) or to apeptide of NY-ESO-1 (New York esophageal squamous cell carcinoma 1). Twoisotype negative controls (FIG. 1A format and FIG. 1C format) comprisingirrelevant target antigen binding domains were also included.

As illustrated in FIGS. 16A, 16B and 16C, the molecules bound, asexpected, to NY-ESO-1, MAGEA4a, or MAGEA4b expressing cells by flowcytometry.

As illustrated in FIGS. 17A and 17B, the multispecific moleculestargeting two distinct antigens (Molecule A) or two different epitopesof a single antigen (Molecule B) potently induced cytotoxicity of bothmetastatic non-small cell lung cancer (NSCLC) cells (FIG. 17A) and NSCLCcells (FIG. 17B), with the multispecific molecule targeting two distinctantigens (Molecule A) showing increased potency relative to themultispecific molecule targeting two distinct epitopes of the sameantigen (Molecule B). The addition of an anti-PD-1 antibody and aco-stimulatory bispecific EGFR x CD28 antibody further enhanced thepotency of the exemplary multispecific molecules of the presentinvention (FIG. 1F structures). The relative induction of T-cellactivation of these molecules was also evaluated, and is shown in FIGS.17C (in metastatic NSCLC cells) and 17D (in NSCLC cells).

The relative cytotoxic activity and potency of multispecific moleculestargeting one or two antigens (distinct epitopes or distinct antigens),and having the structures of FIGS. 1C and 1F, was compared to thecytotoxicity of a conventionally formatted molecule (FIG. 1A), alone orin combination with an anti-PD-1 antibody and a co-stimulatorybispecific EGFR x CD28 antibody. The positive control and the isotypecontrols were as discussed above in this Example. As illustrated inFIGS. 18A and 18B, the multispecific molecules were more potent than theconventionally formatted molecules, and the multispecific moleculestargeting two distinct epitopes (FIG. 18A) or two distinct antigens(FIG. 18B) were more potent than the multispecific molecules targetingthe same antigen at both target antigen-binding domains. The relativeinduction of T-cell activation of these molecules is shown in FIGS. 18C,18D, 18E and 18F.

Example 10: Relative Cytotoxicity of Multispecific Molecule TargetingTwo Antigens Relative to a Combination of Conventionally FormattedMolecules Targeting the Same Antigens

Cytotoxicity of an exemplary multispecific molecule of the presentinvention (FIG. 1F structure) targeting two different antigens wasmeasured as discussed above, and compared to the cytotoxicity of acombination of conventionally formatted molecules (FIG. 1A structure)targeting the same two antigens, alone or in combination with ananti-PD-1 antibody and a co-stimulatory bispecific EGFR x CD28 antibody.

The cytotoxicity assay targeted MAGEA4 expressing-SCaBER cells(Bladder), and demonstrated that the multispecific molecule targetingboth MAGEA4a and MAGEA4b (which are non-overlapping peptides of MAGEA4)was more potent than the combination of conventionally-formattedbispecific antibodies targeting the same two MAGEA4 peptides, as shownin FIG. 19A. The addition of an anti-PD-1 antibody and a co-stimulatorybispecific EGFR x CD28 antibody further enhanced the potency of theexemplary multispecific molecule of the present invention (FIG. 1Fstructure). The relative induction of T-cell activation by these samemolecules is shown in FIG. 19B.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1-93. (canceled)
 94. A method of treating cancer, comprisingadministering to a subject in need thereof, a multispecificantigen-binding molecule, comprising: (a) a first polypeptidecomprising, from N-terminus to C-terminus (i) a first antigen-bindingdomain that specifically binds a T cell antigen, (ii) a firstmultimerizing domain, and (iii) a second antigen-binding domain thatspecifically binds a T cell antigen; and (b) a second polypeptidecomprising, from N-terminus to C-terminus (i) a third antigen-bindingdomain that specifically binds a target antigen, and (ii) a secondmultimerizing domain, wherein the first and the second multimerizingdomains associate with one another to form the molecule.
 95. The methodof claim 94, wherein the second polypeptide further comprises a fourthantigen-binding domain at the C-terminus of the second multimerizingdomain.
 96. The method of claim 95, wherein the fourth antigen-bindingdomain specifically binds a target antigen.
 97. The method of claim 95,wherein the fourth antigen-binding domain specifically binds a T cellantigen.
 98. The method of claim 94, wherein one or more of theantigen-binding domains is a Fab domain.
 99. The method of claim 94,wherein one or more of the antigen-binding domains is an scFv domain.100. The method of claim 99, wherein the scFv domain comprises a heavychain variable region (HCVR) comprising a cysteine mutation at residue44, and a light chain variable region comprising a cysteine mutation atresidue 100 (Kabat numbering).
 101. The method of claim 99, wherein thescFv is connected to the C-terminus of the first and/or secondmultimerizing domain via a linker of from 5 to 25 amino acids,optionally a (G4S)₃ linker.
 102. The method of claim 94, wherein thefirst antigen-binding domain and the third antigen-binding domain areFab domains.
 103. The method of claim 94, wherein the secondantigen-binding domain is an scFv domain.
 104. The method of claim 95,wherein the fourth antigen-binding domain is an scFv domain.
 105. Themethod of claim 95, wherein the first and third antigen-binding domainsare Fab domains, and the second and fourth antigen-binding domains arescFv domains.
 106. The method of claim 94, wherein the T cell antigenis: (a) CD3; (b) a co-stimulatory molecule or a check-point inhibitor ona T cell; or (c) selected from the group consisting of CD27, CD28, 4-1BBand PD-1.
 107. The method of claim 106, wherein the T cell antigen isCD3, and each antigen-binding domain that specifically binds a T cellantigen comprises a HCVR comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 34, SEQ ID NO: 138, SEQID NO: 154, and sequences with at least 95% sequence identity to any oneof SEQ ID NOs: 2, 34, 138 and 154, and a LCVR comprising the amino acidsequence of SEQ ID NO: 162 or a sequence with at least 95% sequenceidentity to SEQ ID NO:
 162. 108. The method of claim 94, wherein thetarget antigen is a tumor-associated antigen.
 109. The method of claim94, wherein the first and second multimerizing domains areimmunoglobulin Fc domains.
 110. The method of claim 109, wherein thefirst and second multimerizing domains associate with one another viadisulfide bonding.
 111. The method of claim 94, wherein the firstmultimerizing domain and the second multimerizing domain are human IgG1or human IgG4 Fc domains.
 112. The method of claim 109, wherein thefirst multimerizing domain or the second multimerizing domain comprisesan amino acid substitution that reduces affinity for Protein A bindingcompared to a wild-type Fc domain of the same isotype.
 113. The methodof claim 109, wherein the amino acid substitution comprises an H435Rmodification, or H435R and Y436F modifications (EU numbering).
 114. Themethod of claim 113, wherein the first multimerizing domain comprisesthe H435R and Y436F modifications.
 115. The method of claim 94, whereinthe first polypeptide, the second polypeptide, or both the first and thesecond polypeptides comprise a modified hinge domain that reducesbinding affinity for an Fcγ receptor relative to a wild-type hingedomain of the same isotype.
 116. The method of claim 94, wherein thetarget antigen is a peptide in the context of the groove of a majorhistocompatibility complex (MHC) protein.
 117. The method of claim 94,wherein the target antigen is present at a density of from 100 to 5000copies per target cell or from 1000 to 20,000 copies per target cell.118. The method of claim 94, wherein the molecule is administered incombination with a second therapeutic agent comprising a bispecificantigen-binding molecule comprising a first antigen-binding domain thatbinds a target antigen (TA) and a second antigen-binding domain thatbinds a T-cell antigen.
 119. The method of claim 118, wherein the targetantigen is a tumor-cell antigen.
 120. The method of claim 118, whereinthe second therapeutic agent comprises a bispecific anti-TA x anti-CD28antibody.
 121. The method of claim 120, wherein the second therapeuticagent comprises a bispecific anti-EGFR x anti-CD28 antibody.
 122. Themethod of claim 118, wherein the second therapeutic agent comprises anantibody that binds a check-point inhibitor on a T cell.
 123. The methodof claim 122, wherein the second therapeutic agent comprises ananti-PD-1 antibody.